Phospholidines that are Bcl Family Antagonists for Use in Clinical Management of Conditions Caused or Mediated by Senescent Cells and for Treating Cancer

ABSTRACT

This disclosure provides compounds with Bcl inhibitory activity based on a new chemical scaffold. The phospholidine compounds can include a P-phenyl phospholidine moiety which is substituted with an N-aryl or N-heteroaryl group. The P-phenyl phospholidine moiety can be optionally substituted at phosphorus with thio (═S) instead of oxo (═O). A second heteroatom attached to phosphorus can be cyclically linked to the N-substituted nitrogen atom of the phospholidine that is attached to the phosphorus to provide, together with the phosphorus atom through which they are connected, a heterocyclic ring. By incorporating such a cyclic constraint between two phosphorus substituents of the core linking moiety a favorable binding conformation can be promoted in the compounds. Selected compounds promote apoptosis in senescent cells, and can be developed for treating senescent-related conditions, such as osteoarthritis, ophthalmic disease, pulmonary disease, and atherosclerosis. Selected compounds promote apoptosis in cancer cells, and can be developed as chemotherapeutic agents.

RELATED APPLICATIONS

This application is a continuation of international patent applicationPCT/US2019/030028, filed Apr. 30, 2019 (pending), which claims priorityto U.S. provisional patent application nos. 62/664,850 (acylbenzylamines), 62/664,891 (phosphonamidates), 62/664,860(phospholidines), and 62/664,863 (acyl phosphonamidates), all filed Apr.30, 2018. All the aforelisted priority applications are herebyincorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The technology disclosed and claimed below relates generally to thefield of senescent cells and their role in age-related conditions. Inparticular, this disclosure provides new scaffolds for chemicalstructures that inhibit Bcl protein activity.

SUMMARY

The invention provided here creates a new paradigm for the treatment ofsenescence related conditions. The disclosure that follows outlines astrategy for selectively eliminating senescent cells, and provideseffective compounds, pharmaceutical compositions, developmentstrategies, and treatment protocols, and describes many of the ensuingbenefits.

A new family of Bcl inhibitors has been developed based on a newchemical scaffold. Some of the Bcl inhibitors in this family areparticularly effective senolytic agents. Contacting senescent cells invitro or in vivo with the compounds and compositions of this disclosureselectively modulates or eliminates such cells. The inhibitors can beused for administration to a target tissue in a subject having anage-related condition, thereby selectively eliminating senescent cellsin or around the tissue and relieving one or more symptoms or signs ifthe conditions. Selected compounds from the family can be formulated andmarketed as chemotherapeutic agents.

The invention is put forth in the description that follows, in thefigures, and in the appended claims.

DRAWINGS

FIG. 1A to 1C depict a synthesis scheme that is generally useful forpreparing the phospholidine compounds.

FIGS. 2A to 2C depict the synthesis of an exemplary compound anaccordance with this disclosure.

FIGS. 3A and 3B are tables of data showing the ability of exemplaryphospholidine compounds to inhibit Bcl activity and to remove senescentcells.

FIG. 4 is taken from a three-dimensional model in which the Bclinhibitors described in this disclosure are fit into the crystalstructure of Bcl family proteins. The annotations can be used as a guideto the reader for developing additional compounds that fall within theformulas shown above, that would retain Bcl inhibition activity andsenolytic activity.

FIGS. 5A, 5B, and 5C show expression of senescent cell markers p16,IL-6, and MMP13 respectively in an osteoarthritis model. The senescencephenotype can be ameliorated by a senolytic agent.

FIG. 6A shows that an effective senolytic agent restores symmetricalweight bearing to treated mice in the osteoarthritis model. FIGS. 6B,6C, and 6D are images showing histopathology of the joints in thesemice. Treatment with the agent helps prevent or reverses destruction ofthe proteoglycan layer.

FIGS. 7A and 7B show reversal of both neovascularization andvaso-obliteration in the mouse oxygen-induced retinopathy (OIR) modelwhen intravitreally administered with a senolytic agent.

FIGS. 7C and 7D are taken from the streptozotocin (STZ) model fordiabetic retinopathy. STZ-induced vascular leakage is attenuated withthe intravitreal administration of a senolytic agent.

FIG. 8 shows that removing senescent cells helps restore oxygensaturation (SPO₂) in a mouse model for cigarette smoke (CS) induced COPD(chronic obstructive pulmonary disease).

FIG. 9 shows data taken from a mouse model for atherosclerosis, in whichinbred mice lacking the LDL receptor were fed a high-fat diet. The rightpanel shows staining for plaques in the aorta. The middle panel showsquantitatively that the surface area of the aorta covered with plaqueswas reduced by treatment with a senolytic agent.

DETAILED DESCRIPTION

Senescent cells are characterized as cells that no longer havereplicative capacity, but remain in the tissue of origin, eliciting asenescence-associated secretory phenotype (SASP). It is a premise ofthis disclosure that many age-related conditions are mediated bysenescent cells, and that selective removal of the cells from tissues ator around the condition can be used clinically for the treatment of suchconditions.

The technology described and claimed below represents the firstdescription of a new class of Bcl inhibitors that can be used toselectively eliminate senescent cells from a target tissue for purposesof treatment of age-related conditions.

Inhibition of Bcl Protein Activity

The Bcl protein family (TC #1.A.21) includes evolutionarily-conservedproteins that share Bcl-2 homology (BH) domains. Bcl proteins are mostnotable for their ability to up- or downregulate apoptosis, a form ofprogrammed cell death, at the mitochondrion. The following explanationis provided to assist the user in understanding some of the scientificunderpinnings of the compounds of this disclosure. These concepts arenot needed to practice the invention, nor do they limit the use of thecompounds and methods described here in any manner beyond that which isexpressly stated or required.

In the context of this disclosure, the Bcl proteins of particularinterest are those that downregulate apoptosis. Anti-apoptotic Bclproteins contain BH1 and BH2 domains, some of them contain an additionalN-terminal BH4 domain (Bcl-2, Bcl-x(L) and Bcl-w (Bcl-2L2), Inhibitingthese proteins increases the rate or susceptibility of cells toapoptosis. Thus, an inhibitor of such proteins can be used to helpeliminate cells in which the proteins are expressed.

In the mid-2000s, Abbott Laboratories developed a novel inhibitor ofBcl-2, Bcl-xL and Bcl-w, known as ABT-737 (Navitoclax). This compound ispart of a group of BH3 mimetic small molecule inhibitors (SMI) thattarget these Bcl-2 family proteins, but not Al or Mcl-1. ABT-737 issuperior to previous BCL-2 inhibitors given its higher affinity forBcl-2, Bcl-xL and Bcl-w. In vitro studies showed that primary cells frompatients with B-cell malignancies are sensitive to ABT-737. In humanpatients, ABT-737 is effective against some types of cancer cells, butis subject to dose-limiting thrombocytopenia.

US 2016/0339019 A1 (Laberge et al.) describes treatment of certainage-related conditions using MDM2 inhibitors, Bcl inhibitors, and Aktinhibitors. US 20170266211 A1 (David et al.) describes the use ofparticular Bcl inhibitors for treatment of age-related conditions. U.S.Pat. Nos. 8,691,184, 9,096,625, and 9,403,856 (Wang et al.) describe Bclinhibitors in a small-molecule library.

It has now been discovered that compounds based on the new scaffolddescribed here fits into the active site of Bcl protein to providestrong Bcl inhibition and/or promote apoptosis of target cells. Thesecompounds can be developed as highly potent and specific drugs to targetsenescent cells and cancer cells, as described in the sections thatfollow.

Phospholidine Inhibitor Compounds

This disclosure includes Bcl inhibitor phospholidine compounds having ascaffold based on a core N-substituted phospholidine linking moiety thatcan provide for a favorable binding conformation in the active site of aBcl protein and gives compounds that have potent inhibition activityand/or promote apoptosis of target cells. The N-substitutedphospholidine linking moiety can link a N-aryl or N-heteroarylsubstituent with a P-aryl or P-heteroaryl substituent via a core cyclicgroup that includes a N—P(═X) group where X is O or S.

Phospholidine compounds of the disclosure can include a P-phenylphospholidine moiety which is substituted with an N-aryl or N-heteroarylgroup. The P-phenyl phospholidine moiety can be optionally substitutedat phosphorus with thio (═S) instead of oxo (═O). In the core cyclicphospholidine group, the phosphorus can be further connected with asulfur, oxygen or second nitrogen ring atom. Alternatively, thephosphorus can be linked to a saturated carbon atom. As such, thephospholidine compound may include a P-phenyl oxazaphospholidine group,a P-phenyl thiazaphospholidine group or a P-phenyl imidazaphospholidinegroup. The phosphorus atom of the core N-substituted phospholidinelinking moiety is tetrahedral and can be chiral. As such, thephospholidine compounds of this disclosure can be present as astereoisomer.

A second heteroatom attached to phosphorus can be cyclically linked tothe N-substituted nitrogen atom of the phospholidine that is attached tothe phosphorus to provide, together with the phosphorus atom throughwhich they are connected, a heterocyclic ring. By incorporating such acyclic constraint between two phosphorus substituents of the corelinking moiety a favorable binding conformation can be promoted in thecompounds.

The phenyl ring of the P-phenyl phospholidine moiety is furthersubstituted to provide a desirable configuration of substituents thatcan fit into particular locations of the active site of Bcl protein. Thenitrogen of the P-phenyl phospholidine moiety is substituted with anN-aryl or N-heteroaryl group which is itself further substituted toprovide a desirable configuration of substituents that fit intoparticular locations of the active site of Bcl protein. The N-aryl orN-heteroaryl group can be N-phenyl which phenyl ring is furthersubstituted.

This technology includes a compound described by Formula (I):

where:

-   -   X¹ is O or S;    -   R¹ is selected from R²¹, SR²¹, OR²¹, and NR²¹R²²;    -   R³ is selected from hydrogen, alkyl, substituted alkyl,        alkanoyl, substituted alkanoyl, alkoxycarbonyl and substituted        alkoxycarbonyl; and/or R¹ and R³ together with the N and P atoms        through which they are connected form a 5-, 6- or 7-membered        heterocyclic ring, optionally substituted with one or more R²³;    -   R⁴ is selected from hydrogen, alkyl, substituted alkyl, nitro,        alkylsulfonyl, substituted alkylsulfonyl, alkylsulfinyl,        substituted alkylsulfinyl, cyano, alkylcarbonyl, substituted        alkylcarbonyl, C(O)OH, C(O)NH₂, halogen, SO₂NH₂,        alkylaminosulfonyl, substituted alkylaminosulfonyl,        alkylsulfonylamino and substituted alkylsulfonylamino, alkanoyl,        substituted alkanoyl, alkylaminocarbonyl, substituted        alkylaminocarbonyl, alkyloxycarbonyl and substituted        alkyloxycarbonyl;    -   R²¹ and R²² are independently selected from hydrogen, alkyl and        substituted alkyl; or R²¹ and R²² together with the N atom        through which they are connected form a 5- or 6-membered        heterocyclic ring, optionally substituted with one or more R²³;    -   each R²³ is independently selected from alkyl, substituted        alkyl, —CONH₂, COOH, CONHR²¹, hydroxyl, halogen, alkoxy and        substituted alkoxy;    -   Z² is selected from —NR⁵R⁶, hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkoxy, substituted alkoxy,        alkylsulfanyl, substituted alkylsulfanyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, carbocycle, substituted carbocycle, heterocycle,        substituted heterocycle, arylalkoxy, substituted arylalkoxy,        aryloxy, substituted aryloxy, aryloxyalkoxy, substituted        aryloxyalkoxy, arylsulfanyl, substituted arylsulfanyl,        arylsulfanylalkoxy, substituted arylsulfanylalkoxy,        cycloalkylalkoxy, substituted cycloalkylalkoxy, cycloalkyloxy,        substituted cycloalkyloxy, halogen, carbonyloxy, haloalkoxy,        haloalkyl, hydroxy and nitro;    -   Z³ is selected from heterocycle, substituted heterocycle,        —NR⁵R⁶, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, carbocycle and substituted carbocycle;    -   R⁵ and R⁶ are independently selected from hydrogen, alkyl and        substituted alkyl;    -   R¹¹ and R¹² are each one or more optional substituents each        independently selected from alkyl, substituted alkyl, alkoxy,        substituted alkoxy, halogen, cyano, nitro, carboxy, C(O)NH₂,        SO₂NH₂, sulfonate, hydroxyl, alkylsulfonyl, substituted        alkylsulfonyl, alkylaminosulfonyl, substituted        alkylaminosulfonyl, alkylsulfonylamino, substituted        alkylsulfonylamino, alkyloxycarbonyl, substituted        alkyloxycarbonyl and —NR⁵R⁶; and    -   R³¹ is selected from H, R¹² and L³-Y³ wherein L³ is a linker and        Y³ is selected from aryl, substituted aryl, heteroaryl and        substituted heteroaryl.

This disclosure includes phospholidine compounds with R¹ and R³ beingcyclically linked to provide a heterocycle ring. Phospholidine compoundsof Formula (I) include R¹ and R³ being connected to provide a 5-, 6- or7-membered heterocyclic ring together with the N and P atoms throughwhich they are connected. In addition to the N and P atoms, thisheterocyclic ring can further include an O, S or a second N ring atom.This disclosure includes R¹ being R²¹, SR²¹, OR²¹ or NR²¹R²² where R²¹and R³ together with the atoms through which they are connected form a5-, 6- or 7-membered heterocyclic ring, optionally substituted with oneor more R²³. For example, R¹ and R³ can be cyclically linked (—R¹*R³—)to provide a —X²(CH₂)_(p)— linker between the phosphorus atom and theadjacent nitrogen atom, where p is 2 or 3 and X² is O, S or NH.Optionally, the heterocyclic ring can include a carbonyl group as partof the ring, and the heterocyclic ring can be optionally substituted,e.g., with one or more R²³ groups.

As such, the phospholidine compound of Formula (I) can be furtherdescribed by Formula (II):

where:

-   -   X² is selected from Z¹², S, O, and NR²²;    -   Z¹¹ is selected from —C(═O)—, —C(═O)X³— and Z¹²;    -   each Z¹² is independently CR²⁴R²⁵;    -   X³ is O or S;    -   n is 0, 1, 2 or 3; and    -   each R²⁴ and each R²⁵ is independently selected from hydrogen,        alkyl and substituted alkyl.

This disclosure includes X¹ being O. Alternatively, X¹ can be S. InFormula (II), Z¹¹ can be Z¹². Sometimes, Z¹¹ can be a carbonyl (C═O) orcarbonyloxy (—C(═O)O—). When Z¹¹ is selected from —C(═O)X³— n is 0, 1 or2. When Z¹² is —C(═O)— or Z¹² n is 1, 2 or 3. This disclosure includesX² being NH or NR²². R²² can be alkyl or substituted alkyl. Optionally,R²² is C₁₋₆ alkyl, such as methyl. This disclosure includes X² beingZ¹². This disclosure includes X² being O. X² can also be S. Sometimes,every Z¹² of the ring is CH₂. Optionally, one or more Z¹² groups of thering can be substituted with a R²⁴ and/or R²⁵ substituent.

This disclosure includes compounds of formula (I) or (II) where the 5-,6- or 7-membered heterocyclic ring connecting the N and P atoms includesa sidechain substituent that derives from an amino acid residue. Forexample, one of R²⁴ and R²⁵ of a Z¹² group can be a group correspondingto an amino acid sidechain. For each Z¹² group, one of R²⁴ and R²⁵ canbe a sidechain group of an amino acid residue such as alanine, valine,leucine, isoleucine, glutamine, glutamic acid, aspartic acid,asparagine, serine, threonine, cysteine, methionine, lysine, arginine,ornithine, phenylalanine, tyrosine, tryptophan or histidine, or asubstituted version thereof. In general, the heterocyclic ring includesonly one Z¹² group that includes an amino acid derived sidechain, wherethe remaining Z¹² groups R²⁴ and R²⁵ are each hydrogen.

As such, Z¹² can include a chiral center having a chiralitycorresponding to an L-amino acid residue. Alternatively, Z¹² can have achirality corresponding to a D-amino acid residue. Certain moieties ofan amino acid synthon (e.g., the alpha-amino group or the backbonecarboxylic acid group) may be modified or removed upon incorporationinto the heterocyclic ring of the compound, while still providing forretention or incorporation of an amino acid-derived sidechain group ofinterest.

A stereoisomer of a phospholidine compound includes a chiral phosphorusstereocenter. Any of the formula or structures of this disclosure caninclude stereoisomer(s) at the tetrahedral phosphorus stereocenter ofthe phospholidine moiety. As such, the phospholidine compound of Formula(II) can be enriched in a stereoisomer of Formula (IIa) or (IIb):

This disclosure includes an optically pure compound of Formula (IIa).This disclosure includes an optically pure compound of Formula (IIb).

In the core N-substituted phospholidine linking moiety of any of theformulas of this disclosure (e.g., Formulas (I)-(VIIb)), R³ and R¹ canbe cyclically linked via —Z¹¹—(Z¹²)_(n)—X²— as shown in Formula (II)where together with the N and P atoms through which they are connectedprovide a phosphorus-containing heterocyclic ring. This ring can be 5-or 6-membered and optionally substituted with one or more groupsindependently selected from alkyl, substituted alkyl, alkoxy,substituted alkoxy, halogen, cyano, nitro, carboxy, C(O)NH₂, SO₂NH₂,sulfonate, hydroxyl, alkylsulfonyl, substituted alkylsulfonyl,alkylaminosulfonyl, substituted alkylaminosulfonyl, alkylsulfonylaminoand substituted alkylsulfonylamino, alkoxycarbonyl, substitutedalkoxycarbonyl, alkylamino, dialkylamino, substituted alkylamino andsubstituted dialkylamino.

The phospholidine compound of Formula (I) or (II) can be furtherdescribed by one of Formulas (IIIa)-(IIIh):

-   -   where R²⁴ and R²⁵ are independently selected from H, alkyl, and        substituted alkyl, and r is 1 or 2. Sometimes r is 1.        Alternatively r is 2. This disclosure includes compounds of        Formula (IIIa) to (IIIh) with X¹ being O. Optionally, X¹ can be        S.

The phospholidine compound of Formula (IIIa) can be further described byone of Formulas (IIIa1)-(IIIa2):

-   -   where R^(25a) is selected from alkyl, and substituted alkyl.

In the core phospholidine linking moiety of any of the formulas of thisdisclosure (e.g., Formulas (I)-(VIIb)), R⁴ can be nitro, alkylsulfonylor substituted alkylsulfonyl. Sometimes, R⁴ is nitro. Alternatively, R⁴can be an alkylsulfonyl. Optionally, R⁴ is CH₃SO₂—. This disclosureincludes compounds and formula where R⁴ can be a substitutedalkylsulfonyl. Optionally, R⁴ is CF₃SO₂—. R⁴ can also be cyano.Optionally, R⁴ can be alkylcarbonyl, substituted alkylcarbonyl, —C(O)OHor —C(O)NH₂.

This disclosure includes R⁴ being alkylaminosulfonyl or substitutedalkylaminosulfonyl. Also included is R⁴ being alkoxycarbonyl orsubstituted alkoxycarbonyl. Also included is R⁴ being alkylsulfonylaminoor substituted alkylsulfonylamino. Alternatively, R⁴ can be alkyl orsubstituted alkyl.

In any of the formulas of this disclosure, Z² can be —NR⁵R⁶ where R⁵ andR⁶ are independently selected from hydrogen, alkyl and alkyl substitutedwith one or more groups selected from arylsulfanylalkyl,heteroarylsulfanylalkyl, alkenyl, alkoxyalkyl, alkoxycarbonylalkyl,alkyl, alkylsulfonylalkyl, aryl, arylalkyl, arylsulfonylalkyl,aryloxyalkyl, arylsulfinylalkyl, arylsulfonylalkyl, heteroaryl,heteroarylalkyl, heteroarylsulfonylalkyl, heteroaryloxyalkyl,heteroarylsulfinylalkyl, heteroarylsulfonylalkyl, carboxyalkyl,cycloalkenyl, cycloalkenylalkyl, cycloalkyl, (cycloalkyl)alkyl,cycloalkylcarbonyl, (heterocycle)sulfanylalkyl, hydroxyalkyl,alkylamino, dialkylamino and heterocycle (e.g., piperidinyl, piperazinylor morpholinyl), wherein said one or more groups are optionally furthersubstituted. This disclosure includes Z² being —NR⁵R⁶ where R⁵ ishydrogen. R⁶ can be arylsulfanylalkyl, heteroarylsulfanylalkyl,aryloxyalkyl or heteroaryloxyalkyl, which R⁶ is optionally furthersubstituted, e.g., substituted with a heterocycle or substitutedheterocycle. The heterocycle or substituted heterocycle can be asubstituent of an alkyl moiety of R⁶ and in some cases is a piperidinyl,piperazinyl or morpholinyl heterocycle.

In the formulas of this disclosure, Z³ can be a substituted heterocycle.Z³ can be a monocyclic 6-membered saturated heterocycle that is furthersubstituted. This disclosure includes Z³ being a substitutedpiperidinyl. Optionally, Z³ can be a substituted piperazinyl. The Z³heterocycle group can be substituted with a group including three linkedcyclic groups (e.g., three aryl, heteroaryl, carbocycle and/orheterocycle) connected via optional linkers to each other and in alinear fashion to first heterocycle of Z³. Z³ can be piperidinyl orpiperazinyl substituted with a tri-(arylene and/or heteroarylene) group.Optionally, Z³ can be piperidinyl or piperazinyl substituted with atri-arylene, tri-heteroarylene, aryl-heteroaryl-aryl,heteroaryl-heteroaryl-aryl, aryl-heteroaryl-heteroaryl,aryl-cycloalkyl-aryl or aryl-heterocycle-aryl, where each ring of saidgroup is optionally further substituted.

Alternatively, in the formulas of this disclosure, Z³ can be asubstituted carbocycle. Z³ can be a monocyclic 6-membered saturatedcarbocyclic that is further substituted. The Z³ carbocycle group can besubstituted with a group including three linked cyclic groups (e.g.,three aryl, heteroaryl, carbocycle and/or heterocycle) connected viaoptional linkers to each other and in a linear fashion to the firstcarbocycle of Z³. Z³ can be cyclohexyl substituted with a tri-arylene,tri-heteroarylene, aryl-heteroaryl-aryl, heteroaryl-heteroaryl-aryl,aryl-heteroaryl-heteroaryl, aryl-cycloalkyl-aryl oraryl-heterocycle-aryl, where each ring of said group is optionallyfurther substituted.

This disclosure includes compounds having a Y³ aryl or heteroaryl groupthat is capable of pi stacking interactions with the phenyl of theP-phenyl phospholidine moiety in the core structure. Pi stacking (or π-πstacking) interactions refer to attractive, noncovalent interactionsbetween aromatic rings. This disclosure includes compounds of Formula(I) where R³¹ is L³-Y³ where L³ is a linker and Y³ is aryl, substitutedaryl, heteroaryl or substituted heteroaryl. Alternatively, a Y³ groupcapable of similar pi stacking interactions can be linked (via L³) tothe Z² substituent group. The linker L³ can provide for a configurationof the Y³ group adjacent to the phenyl ring of the P-phenylphospholidine moiety such that the two aromatic groups are capable ofintramolecular pi stacking interactions.

As such, the phospholidine compound of Formula (I) can be furtherdescribed by Formula (IVa) or Formula (IVb):

where:

-   -   Z⁴ is selected from CH, CR¹³ and N;    -   L², L³, L⁴ and L⁵ are each independently a linker;    -   Y² is selected from alkyl, substituted alkyl, hydroxyl, alkoxy,        substituted alkoxy, —NR⁷R⁸, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, carbocycle, substituted carbocycle,        heterocycle and substituted heterocycle;    -   Y³ is selected from aryl, substituted aryl, heteroaryl and        substituted heteroaryl;    -   Y⁴, Y⁵ and Y⁶ are independently selected from aryl, substituted        aryl, heteroaryl, substituted heteroaryl, carbocycle,        substituted carbocycle, heterocycle, and substituted        heterocycle;    -   R⁷ and R⁸ are independently selected from hydrogen, alkyl and        substituted alkyl, or R⁷ and    -   R⁸ together with the nitrogen to which they are attached form a        5-, 6- or 7-membered heterocyclic ring or substituted 5-, 6- or        7-membered heterocyclic ring; and    -   R¹³ is one or more optional substituents selected from alkyl,        substituted alkyl, alkoxy, substituted alkoxy, halogen, cyano,        nitro, carboxy, C(O)NH₂, SO₂NH₂, sulfonate, hydroxyl,        alkylsulfonyl, substituted alkylsulfonyl, alkylaminosulfonyl,        substituted alkylaminosulfonyl, alkylsulfonylamino, substituted        alkylsulfonylamino, alkyloxycarbonyl, substituted        alkyloxycarbonyl, alkylamino, dialkylamino, substituted        alkylamino and substituted dialkylamino.

This disclosure includes compounds of Formula (IVa), where Y³ is asubstituted or unsubstituted fused bicyclic heteroaryl. In Formula(IVa), Y³ can be a pyrrolo-pyridine, optionally further substituted.This disclosure includes compounds of Formula (IVb), where L² is atrivalent linker connecting Y² and Y³ to a nitrogen N, and Y³ is asubstituted or unsubstituted phenyl. The compounds of Formulas (IVa) and(IVb) can include a Y² group selected from OR and OP(═O)(OR)₂, whereeach R is independently H, alkyl or substituted alkyl. Each R can be aC₁₋₆ alkyl such as ethyl or tert-butyl. Optionally, Y² can be NR⁷R⁸,where Wand R⁸ together with the nitrogen to which they are attached canform a 5- or 6-membered heterocyclic ring, optionally furthersubstituted. Where Y² is NR⁷R⁸, the 5- or 6-membered heterocyclic ringis optionally substituted on carbon with one or more R⁴⁰, and if the 5-or 6-membered heterocyclic ring contains a second nitrogen, that secondnitrogen is optionally substituted with R⁴⁰* to form a tertiary amine.Each R⁴⁰*, if present, can be selected from alkyl, substituted alkyl,—(CH₂)_(m1)OR and —(CH₂)_(m2)OP(═O)(OR)₂, where m1 and m2 areindependently an integer from 1 to 6 and each R is independently H,alkyl or substituted alkyl. R can be a C₁₋₄alkyl such as ethyl ortert-butyl. Each R⁴⁰, if present, is independently selected from —OR,—N(R)₂, —(CH₂)_(m1)OR, —(CH₂)_(m3)N(R)₂, —(CH₂)_(m2)OP(═O)(OR)₂,—OP(═O)(OH)₂, and —OP(═O)(OR)₂ where m1, m2 and m3 are eachindependently an integer from 1 to 6 and each R is independently H,alkyl or substituted alkyl. R can be a C₁₋₄alkyl such as ethyl ortert-butyl.

As described above, Z³ can be a substituted heterocycle, such as asubstituted piperidinyl or substituted piperazinyl. The Z³ heterocyclegroup can be substituted with a group including three linked cyclicgroups (e.g., three aryl, heteroaryl, carbocycle and/or heterocycle)connected via optional linkers to each other and in a linear fashion toZ³. The disclosure includes compounds where the Z³ substituent is atriheteroaryl, aryl-heterocycle-aryl, aryl-heteroaryl-aryl,heteroaryl-heteroaryl-aryl or aryl-heteroaryl-heteroaryl, where eachring of the Z³ substituent is optionally further substituted. As such,one substituent of such a Z³ substituted heterocycle can be described bythe formula Y⁶-L⁵-Y⁵-L⁴-Y⁴— where Y⁴ and Y⁵ are each independently aryl,substituted aryl, heteroaryl, substituted heteroaryl, carbocycle,substituted carbocycle, heterocycle or substituted heterocycle and L⁴and L⁵ are optional linkers. This disclosure includes L⁴ and L⁵ beingsingle covalent bonds. The compounds may include Y⁴ and Y⁵ groups thatare monocyclic aromatic or non-aromatic 5-, 6- or 7-membered ringslinked in sequence to the first heterocyclic group of Z³. Thisdisclosure includes a terminal Y⁶ group being phenyl or substitutedphenyl. The Y⁴ group can be linked via a 1,2- or 1,3-configuration.

This disclosure also includes Y⁴ being phenyl or substituted phenyl.Optionally, Y⁴ is a 1,3-phenylene or substituted 1,3-phenylene. Thecompounds may include Y⁵ being a heterocycle or substituted heterocycle.Optionally, Y⁵ is a heterocycle or substituted heterocycle. Y⁵ can be a5-membered monocyclic heteroaryl, optionally further substituted. Thisdisclosure includes Y⁵ being pyrrole, substituted pyrrole, furan,substituted furan, thiophene or substituted thiophene. When Y⁵ is a5-membered monocyclic heteroaryl, it can be linked to the adjacentgroups (e.g., Y⁶-L⁵- and -L⁴-Y⁴) with a cis-configuration, such as a1,2- or 2,3-configuration. As such, this disclosure includes Y⁵ being anoptionally substituted 2,3-linked pyrrole.

The compounds of Formula (IVa) and Formula (IVb) can include an L³linker being a C₁₋₆alkylene or substituted C₁₋₆alkylene. In Formula(IVb) L² can be a trivalent linker. L² can be a C₁₋₆ alkyl linker orsubstituted C₁₋₆alkyl linker that connects Y² to the nitrogen N and alsoincludes a branching atom that connects Y³-L³ to the nitrogen N. Thebranching atom can be a carbon or a nitrogen atom. This disclosureincludes compounds where L³ and L² together provide a branched linkerconnecting Y² and Y³ to the nitrogen N, where the branched linker is asubstituted C₁₋₁₂ alkylene, such as a C₁₋₆ alkyl linker where one ormore of the backbone carbon atoms are optionally replaced with aheteroatom (e.g., O, S or N).

The compounds of Formula (IVa) and Formula (IVb) can include a Y⁶ groupthat is independently phenyl or substituted phenyl and a Y⁴ that isselected from phenyl or substituted phenyl, e.g., an optionallysubstituted 1,3-linked phenylene. The L⁴ linker can be a covalent bond.Sometimes, the L⁴ linker has one backbone atom. This disclosure alsoincludes L⁵ linkers being a covalent bond, C₁₋₃ alkyl linker,substituted C₁₋₃alkyl linker, O, NR, NH, S, S(═O), SO₂ or C(═O).Optionally, L⁵ can be a single covalent bond.

The compound of Formula (IVb) can thus be further described by Formula(V):

where:

-   -   Y² is selected from OR″, OP(═O)(OR″)₂ and NR⁷R⁸;    -   Z⁵ is selected from NR¹⁹, NH, O and S;    -   Z⁷ is selected from S and O;    -   L⁶ and L⁷ are independently a covalent bond, C₁₋₆alkyl linker or        substituted C₁₋₆alkyl linker;    -   R¹³ and R¹⁴ and each R¹⁵ are independently one or more optional        substituents selected from alkyl, substituted alkyl (e.g., CF₃),        alkoxy, substituted alkoxy (e.g., —OCF₃), halogen, cyano, nitro,        carboxy, C(O)NH₂, SO₂NH₂, sulfonate, hydroxyl, alkylsulfonyl,        substituted alkylsulfonyl, alkylaminosulfonyl, substituted        alkylaminosulfonyl, alkylsulfonylamino, substituted        alkylsulfonylamino, alkyloxycarbonyl, substituted        alkyloxycarbonyl, alkylamino, dialkylamino, substituted        alkylamino and substituted dialkylamino;    -   R¹⁶ is selected from hydrogen, halogen and R¹⁵;    -   R¹⁷ and R¹⁸ are independently selected from hydrogen, cyano,        nitro, halogen, alkyl, substituted alkyl, cycloalkyl,        substituted cycloalkyl, alkenyl, substituted alkenyl,        cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted        alkynyl, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, heterocycle, substituted heterocycle, OR, SR, NRR′,        COR, OCOR, CO₂R, CONRR′, CONRSO₂R′, —C₁₋₃alkyleneCH(OH)CH₂OH,        SO₂R and SO₂NRR′; and    -   R¹⁹ is selected from hydrogen, alkyl, substituted alkyl,        cycloalkyl, substituted cycloalkyl, alkenyl, substituted        alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, heterocycle, substituted heterocycle,        OR, SR, NRR′, COR, OCOR, CO₂R, CONRR′, CONRSO₂R′, NRCOR,        NRCONRR′, NRC(═S)NRR′, NRSO₂R and SO₂NRR′;    -   each R″ is independently H, alkyl or substituted alkyl (e.g., a        C₁₋₆ alkyl such as ethyl or tert-butyl); and    -   R and R′ are independently selected from hydrogen, alkyl,        substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,        substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,        alkynyl, substituted alkynyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, heterocycle, substituted        heterocycle, heterocycle-alkyl- and substituted        heterocycle-alkyl-.

Z⁴ can be linked to an adjacent Y⁴ group via a covalent bond. Thisdisclosure includes compounds of Formula (V), where Z⁴ can be CH orCR¹³. This disclosure includes compounds of Formula (V), where Z⁴ can beN.

This disclosure also includes Y⁵ groups being pyrrole or substitutedpyrrole. The pyrrole can be a 2,3-linked pyrrole. As such, in formula(V), Z⁵ can be NH or NR¹⁹. This disclosure includes L⁵ being a singlecovalent bond, such that Y⁵ (e.g., a 2,3-linked pyrrole) is directlylinked to a terminal phenyl ring.

The compounds of Formula (V) can include L⁶ and L⁷ linkers beingindependently a C₁₋₆alkylene or substituted C₁₋₆alkylene connected via achiral center. This disclosure includes compounds where L⁶ is a covalentbond or a C₁₋₆alkyl linker (e.g., a C₁₋₃ alkylene) where one or more ofthe backbone carbon atoms are optionally replaced with a heteroatom(e.g., O, S or N). Also included are compounds where L⁷ is a covalentbond or a C₁₋₆alkyl linker (e.g., a C₁₋₃ alkylene) where one or more ofthe backbone carbon atoms are optionally replaced with a heteroatom(e.g., O, S or N).

This disclosure includes compounds of Formula (V) where Y² is NR⁷R⁸ andWand R⁸ together with the nitrogen to which they are attached can form a5- or 6-membered heterocyclic ring, optionally further substituted withone or more —OR, —N(R)₂, —(CH₂)_(m1)OR, —(CH₂)_(m3)N(R)₂,—(CH₂)_(m2)OP(═O)(OR)₂, —OP(═O)(OH)₂, and —OP(═O)(OR)₂ where m1, m2 andm3 are each independently an integer from 1 to 6 and each R isindependently H, alkyl or substituted alkyl. R can be a C₁₋₄alkyl suchas methyl, ethyl or tert-butyl. The 5- or 6-membered heterocyclic ringformed by Wand R⁸ can be a 6-membered ring such as morpholinyl,piperidinyl or piperazinyl that is optionally further substituted.

The compound of Formula (V) can be further described by Formula (VIa):

where:

-   -   Z⁶ is selected from O, NR⁴⁰*, CHR⁴⁰, C(R⁴⁰)₂ and CH₂;    -   R⁴⁰* is selected from alkyl, substituted alkyl, —(CH₂)_(m1)OR        and —(CH₂)_(m2)OP(═O)(OR)₂, wherein m1 and m2 are independently        an integer from 1 to 6 and each R is independently H, alkyl or        substituted alkyl (e.g., a C₁₋₄ alkyl such as ethyl or        tert-butyl);    -   each R⁴⁰ is independently selected from —OR, —N(R)₂, —C(O)OR,        —(CH₂)_(m1)OR, —(CH₂)_(m3)N(R)₂, —(CH₂)_(m2)OP(═O)(OR)₂,        —OP(═O)(OH)₂, and —OP(═O)(OR)₂ wherein m1, m2 and m3 are each        independently an integer from 1 to 6 and each R is independently        H, alkyl or substituted alkyl (e.g., a C₁₋₄ alkyl such as ethyl        or tert-butyl);    -   q₁ and q₂ are independently an integer from 1 to 6,    -   or a stereoisomer thereof.

The compound of Formula (V) can be further described by Formula (VIb):

-   -   where Y² is selected from OH, OR, NH₂, NHR, NR₂, —OP(═O)(OR)₂        and —OP(═O)(OH)₂ where each R is independently C₁₋₆ alkyl or        substituted C₁₋₆ alkyl; and q₁ and q₂ are independently an        integer from 1 to 6.

Any of the Formula (I), (IVa), (IVb), (V), (VIa) and (VIb) can have acore phospholidine linking moiety as shown in Formula (II). Thisdisclosure includes such compounds where X¹ is O. Optionally, X¹ is S.Sometimes, the compound is a stereoisomer of Formula (IIa) or (IIb).

Compounds of the Formulas (V) and (VIa) can include stereoisomers ofcompounds having a chiral center at the branching point of linker L². Assuch, the compound of Formulas (V) and (VIa) can be further described byFormula (VIIa):

where:

-   -   X² is selected from Z¹², S, O, and NR²²;    -   Z¹¹ is selected from —C(═O)—, —C(═O)X³— and Z¹²;    -   each Z¹² is independently CR²⁴R²⁵;    -   X³ is O or S;    -   n is 0, 1, 2 or 3;    -   each R²⁴ and each R²⁵ are independently selected from hydrogen,        alkyl and substituted alkyl;    -   R⁴ is selected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹;    -   Z⁴ is selected from CH and N;    -   Z⁶ is selected from O, CHC(O)R¹⁸, and CH(CH₂)_(p)R¹⁸ wherein p        is 0-6 and each R¹⁸ is independently —OR, —N(R)₂, —OP(═O)(OH)₂,        and —OP(═O)(OR)₂ wherein each R is independently H, alkyl or        substituted alkyl (e.g., a C₁₋₄ alkyl such as ethyl or        tert-butyl);    -   R¹⁴ and R¹⁶ are independently hydrogen or halogen; and    -   R¹⁷ is selected from SO₂R⁵², COR⁵², CO₂R⁵², CONR⁵¹R⁵²,        CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²;    -   R¹⁸ and R¹⁹ are independently selected from hydrogen, alkyl and        substituted alkyl;    -   R⁵¹ is selected from C₁₋₆alkyl and substituted C₁₋₆ alkyl; and    -   R⁵² is selected from hydrogen, C₁₋₆alkyl and substituted        C₁₋₆alkyl.

This disclosure includes compounds of Formula (VIIa) as defined by thefollowing compounds 1-18 of Table 1. For any of the compounds of Table1, X¹ can be O. Alternatively, X¹ can be S.

TABLE 1 Compounds of Formula (VIIa) Com- pound X² Z¹¹-(Z¹²)_(n) R⁴ R¹⁴R¹⁶ R¹⁷ R¹⁸ R¹⁹ Z⁴ Z⁶ 1 O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOH2 O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 3 O CH₂CH₂SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 4 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃CH(CH₃)₂ N CHOPO(OH)₂ 5 O CH₂CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH6 O CH₂CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 7 O CH₂CH₂CH₂SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 8 O CH₂CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃CH(CH₃)₂ N CHOPO(OH)₂ 9 O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 10O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 11 O CH₂CH₂ SO₂CH₃ FCl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOH 12 O CH₂CH₂ SO₂CH₃ F Cl SO₂CH₃ CH₃CH(CH₃)₂ N CHOPO(OH)₂ 13 O CH₂CH₂ NO₂ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOH 14O CH₂CH₂ NO₂ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 15 O CH₂CH₂ NO₂ F ClCO₂H CH₃ CH(CH₃)₂ N CHOH 16 O CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ NCHOPO(OH)₂ 17 O CH₂CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 18 OCH₂CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂

Compounds of the Formulas (V) and (VIb) can include stereoisomers ofcompounds having a chiral center at the branching point of linker L². Assuch, the compound of Formulas (V) and (VIb) can be further described byFormula (VIIb):

where:

-   -   Y² is selected from —OR⁵², —N(R⁵²)₂, and —OP(═O)(OR⁵²)₂;    -   R⁴ is selected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹;    -   Z⁴ is selected from CH and N;    -   R¹⁴ and R¹⁶ are independently hydrogen or halogen; and    -   R¹⁷ is selected from SO₂R⁵², COR⁵², CO₂R⁵², CONR⁵¹R⁵²,        CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²;    -   R¹⁸ and R¹⁹ are independently selected from hydrogen, alkyl and        substituted alkyl;    -   R⁵¹ is selected from C₁₋₆alkyl and substituted C₁₋₆ alkyl; and    -   R⁵² is selected from hydrogen, C₁₋₆alkyl and substituted        C₁₋₆alkyl.

This disclosure includes compounds of Formula (VIIb) as defined by thefollowing compounds 19-36 of Table 2. For any of the compounds of Table2, X¹ can be O. Alternatively, X¹ can be S.

TABLE 2 Compounds of Formula (VIIb) Compound X² Z¹¹-(Z¹²)n R⁴ R¹⁴ R¹⁶R¹⁷ R¹⁹ R¹⁹ Z⁴ Y² 19 O CH₂CH₂ NO₂ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N N(CH₃)₂ 20O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N N(CH₃)₂ 21 O CH₂CH₂ SO₂CF₃ FCl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 22 O CH₂CH₂ SO₂CF₃ H Cl CO₂H CH₃ CH(CH₃)₂N N(CH₃)₂ 23 O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 24 OCH₂CH₂ SO₂CH₃ H Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 25 O CH₂CH₂ SO₂CH₃ F ClSO₂CH₃ CH₃ CH(CH₃)₂ N N(CH₃)₂ 26 O CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ NN(CH₃)₂ 27 O CH₂CH₂ NO₂ H Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 28 O CH₂CH₂ NO₂F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N OH 29 O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃CH(CH₃)₂ N OH 30 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N OH 31 O CH₂CH₂SO₂CF₃ H Cl CO₂H CH₃ CH(CH₃)₂ N OH 32 O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃CH(CH₃)₂ N OH 33 O CH₂CH₂ SO₂CH₃ H Cl CO₂H CH₃ CH(CH₃)₂ N OH 34 O CH₂CH₂SO₂CH₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N OH 35 O CH₂CH₂ NO₂ F Cl CO₂H CH₃CH(CH₃)₂ N OH 36 O CH₂CH₂ NO₂ H Cl CO₂H CH₃ CH(CH₃)₂ N OH

The compound of Formula (VIIa) can be further described by Formula(VIIIIa):

where:

-   -   R⁴ is selected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹;    -   n is 1 or 2;    -   Z⁶ is selected from O, CHC(O)R¹⁸, and CH(CH₂)_(p)R¹⁸ wherein p        is 0-6 and each R¹⁸ is independently —OR, —N(R)₂, —OP(═O)(OH)₂,        and —OP(═O)(OR)₂ wherein each R is independently H, alkyl or        substituted alkyl (e.g., a C₁₋₄ alkyl such as ethyl or        tert-butyl);    -   R¹⁴ and R¹⁶ are independently hydrogen or halogen; and    -   R¹⁷ is selected from SO₂R⁵², COR⁵², CO₂R⁵², CONR⁵¹R⁵²,        CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²;    -   R⁵¹ is selected from C₁₋₆alkyl and substituted C₁₋₆ alkyl; and    -   R⁵² is selected from hydrogen, C₁₋₆alkyl and substituted        C₁₋₆alkyl.

This disclosure includes compounds of Formula (VIIIIa) as defined by thefollowing compounds of Table 3.

TABLE 3 Compounds of Formula (VIIIa) Compound R⁴ R¹⁴ R¹⁶ R¹⁷ n Z⁶ 37SO₂CF₃ H Cl CO₂H 1 CHOPO(OH)₂ 38 SO₂CF₃ H Cl CO₂H 1 CHCH₂OPO(OH)₂ 39SO₂CF₃ H Cl CO₂H 1 CHOH 40 SO₂CF₃ H Cl CO₂H 1 CHCH₂OH 41 SO₂CF₃ F ClSO₂CH₃ 1 CHC(O)OH 42 SO₂CF₃ F Cl SO₂CH₃ 2 CHOH

The compound of Formula (Vllb) can be further described by Formula(VIIIb):

where:

-   -   Y² is selected from —OR⁵², —N(R⁵²)₂, and —OP(═O)(OR⁵²)₂;    -   R⁴ is selected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹;    -   n is 1 or 2;    -   R¹⁴ and R¹⁶ are independently hydrogen or halogen;    -   R¹⁷ is selected from SO₂R⁵², COR⁵², CO₂R⁵², CONR⁵¹R⁵²,        CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²;    -   R⁵¹ is selected from C₁₋₆alkyl and substituted C₁₋₆ alkyl; and    -   R⁵² is selected from hydrogen, C₁₋₆alkyl and substituted        C₁₋₆alkyl.

This disclosure includes compounds of Formula (VIIIb) as defined by thefollowing compounds of Table 4.

TABLE 4 Compounds of Formula (VIIIb) Compound R⁴ R¹⁴ R¹⁶ R¹⁷ n Y² 19 NO₂F Cl SO₂CH₃ 1 N(CH₃)₂

This disclosure includes compounds with R¹⁶ being hydrogen, halogen,C₁₋₆ alkyl, substituted C₁₋₆ alkyl (e.g., CF₃) or CN. In Formulas (V),(VIa), (VIb), (VIIa), (VIIb), (VIIIIa) and (VIIIb) R¹⁶ can be halogen.Optionally, R¹⁶ can be chloro. This disclosure includes compounds whereR¹⁶ is not hydrogen. This disclosure includes compounds where R¹⁴ isabsent. Alternatively, in Formulas (V), (VIa), (VIb), (VIIa), (VIIb),(VIIIIa) and (VIIIb) R¹⁴ can be one halogen substituent. Optionally, R¹⁴can be fluoro.

This disclosure includes compounds where R¹⁷ is SO₂R⁵², CO₂R⁵² or COR⁵².Alternatively, R¹⁷ can be CONR⁵¹R⁵², CONR⁵²SO₂R⁵¹ or SO₂NR⁵¹R⁵². R⁵¹ isC₁₋₆ alkyl or substituted C₁₋₆ alkyl and R⁵² is hydrogen, C₁₋₆alkyl orsubstituted C₁₋₆alkyl. In addition, R¹⁸ can be hydrogen. Sometimes, R¹⁸is alkyl. Optionally, R¹⁸ can be substituted alkyl. In addition, R¹⁹ canbe hydrogen. Sometimes, R¹⁹ can be alkyl. Optionally, R¹⁹ can besubstituted alkyl.

The disclosure includes compounds of Formulas (VIIa), (VIIb), (VIIIIa)and (VIIIb) including a stereoisomer of a chiral phosphorusstereocenter. As such, the compound of any one of Formulas(VIIa)-(VIIIb) can have either of the following chirality at phosphorus:

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIa). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. This disclosure includes such compounds where ris 1. Alternatively r is 2. Sometimes, the compound is a stereoisomer ofFormula (IIIa).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIb). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. Sometimes, the compound is a stereoisomer ofFormula (IIIb).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIc). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. This disclosure includes such compounds where ris 1. Alternatively r is 2. Sometimes, the compound is a stereoisomer ofFormula (IIIc).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIId). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. Sometimes, the compound is a stereoisomer ofFormula (IIId).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIe). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. Sometimes, the compound is a stereoisomer ofFormula (IIIe).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIf). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. Sometimes, the compound is a stereoisomer ofFormula (IIIf). Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb),(VIIa) and (VIIb) can have a core phospholidine linking moiety as shownin Formula (IIIg). This disclosure includes such compounds where X¹ isO. Optionally, X¹ is S. This disclosure includes such compounds where ris 1. Alternatively r is 2. Sometimes, the compound is a stereoisomer ofFormula (IIIg).

Any of the Formula (I), (IVa), (IVb), (V), (VIa), (VIb), (VIIa) and(VIIb) can have a core phospholidine linking moiety as shown in Formula(IIIh). This disclosure includes such compounds where X¹ is O.Optionally, X¹ is S. This disclosure includes such compounds where ris 1. Alternatively r is 2. Sometimes, the compound is a stereoisomer ofFormula (IIIh).

This disclosure includes compounds of formula (VIIa) or (VIIb) where the5-, 6- or 7-membered heterocyclic ring connecting the N and P atomsincludes a sidechain substituent that derives from an amino acidresidue. For example, one of R²⁴ and R²⁵ of a Z¹² group can be a groupcorresponding to an amino acid sidechain. For each Z¹² group, one of R²⁴and R²⁵ can be a sidechain group of an amino acid residue such asalanine, valine, leucine, isoleucine, glutamine, glutamic acid, asparticacid, asparagine, serine, threonine, cysteine, methionine, lysine,arginine, ornithine, phenylalanine, tyrosine, tryptophan or histidine,or a substituted version thereof. In general, the heterocyclic ringincludes only one Z¹² group that includes an amino acid derivedsidechain, where the remaining Z¹² groups R²⁴ and R²⁵ are each hydrogen.As such, Z¹² can include a chiral center having a chiralitycorresponding to an L-amino acid residue. Alternatively, Z¹² can have achirality corresponding to a D-amino acid residue.

For any of the formulas and structures depicted herein, such formulasand structures may also include salt forms where the structure providesfor an acidic or basic group. The core phospholidine linking moiety ofthe compounds of this disclosure can be neutral. The compound caninclude a charged moiety (e.g., a basic amino group) at otherlocation(s) of the compound. Where applicable, neutral forms of thegroups are generally depicted in the structures for simplicity, howevervarious salt forms are also meant to be included. A salt of the compoundcould include a monovalent cation salt, such as sodium or potassiumsalt. Other tautomeric arrangements of the groups depicted in theseformulas and structures are possible, and are meant to be included inthis disclosure.

Evaluating Compounds for Senolytic Activity

These and other compounds can be evaluated on the molecular level fortheir ability to perform in a way that indicates they are candidateagents for use according to this disclosure.

For example, where the therapy includes triggering apoptosis ofsenescent cells by way of Bcl-2, Bcl-xL, Bcl-w, or other Bcl familyprotein, compounds can be tested for their ability to inhibit bindingbetween one or more Bcl proteins and their respective cognate ligand.Example 1 provides an illustration of a homogeneous assay (an assay thatdoes not require a separation step) for purposes of determining bindingto the Bcl isoforms. Compounds can be screened on the molecular levelfor their ability to interact with the target isoform, thereby causingsenolysis. Examples 2 and 3 provide illustrations of assays designed forthis purpose.

Alternatively or in addition, compounds can be evaluated for an abilityto kill senescent cells specifically. Cultured cells are contacted withthe compound, and the degree of cytotoxicity or inhibition of the cellsis determined. The ability of the compound to kill or inhibit senescentcells can be compared with the effect of the compound on normal cellsthat are freely dividing at low density, and normal cells that are in aquiescent state at high density. Examples 2 and 3 provide illustrationsof senescent cell killing using the human target tissue fibroblast IMR90cell line and HUVEC cells. Similar protocols are known and can bedeveloped or optimized for testing the ability of the cells to kill orinhibit other senescent cells and other cell types, such as cancercells.

Candidate Bcl inhibitors that are effective in selectively killingsenescent cells in vitro can be further screened in animal models forparticular disease. Examples 4, 5, 6, and 7 of the Experimental Sectionbelow provide illustrations for osteoarthritis, eye disease, lungdisease, and atherosclerosis, respectively.

Formulation of Medicaments

Preparation and formulation of pharmaceutical agents for use accordingto this disclosure can incorporate standard technology, as described,for example, in the current edition of Remington: The Science andPractice of Pharmacy. The formulation will typically be optimized foradministration to the target tissue, for example, by localadministration, in a manner that enhances access of the active agent tothe target senolytic cells and providing the optimal duration of effect,while minimizing side effects or exposure to tissues that are notinvolved in the condition being treated.

Pharmaceutical preparations for use in treating senescence-relatedconditions and other diseases can be prepared by mixing a Bcl inhibitorwith a pharmaceutically acceptable base or carrier and as needed one ormore pharmaceutically acceptable excipients. Depending on the targettissue, it may be appropriate to formulate the pharmaceuticalcomposition for sustained or timed release. Oral timed releaseformulations may include a mixture of isomeric variants, binding agents,or coatings. Injectable time release formulations may include the activeagent in combination with a binding agent, encapsulating agent, ormicroparticle. For treatment of joint diseases such as osteoarthritis,the pharmaceutical composition is typically formulated forintra-articular administration. For treatment of eye disease such asglaucoma, diabetic retinopathy or age-related macular degeneration(AMD), the composition may be formulated for intravitreal orintracameral administration. For treatment of lung diseases, thecomposition may be formulated as an aerosol, or for intratrachealadministration.

This disclosure provides commercial products that are kits that encloseunit doses of one or more of the agents or compositions described inthis disclosure. Such kits typically comprise a pharmaceuticalpreparation in one or more containers. The preparations may be providedas one or more unit doses (either combined or separate). The kit maycontain a device such as a syringe for administration of the agent orcomposition in or around the target tissue of a subject in need thereof.The product may also contain or be accompanied by an informationalpackage insert describing the use and attendant benefits of the drugs intreating the senescent cell associated condition, and optionally anappliance or device for therapeutic delivery of the composition.

Treatment Design

Senescent cells accumulate with age, which is why conditions mediated bysenescent cells occur more frequently in older adults. In addition,different types of stress on pulmonary tissues may promote the emergenceof senescent cells and the phenotype they express. Cell stressorsinclude oxidative stress, metabolic stress, DNA damage (for example, asa result of environmental ultraviolet light exposure or geneticdisorder), oncogene activation, and telomere shortening (resulting, forexample, from hyperproliferation). Tissues that are subject to suchstressors may have a higher prevalence of senescent cells, which in turnmay lead to presentation of certain conditions at an earlier age, or ina more severe form. An inheritable susceptibility to certain conditionssuggests that the accumulation of disease-mediating senescent cells maydirectly or indirectly be influenced by genetic components, which canlead to earlier presentation.

One of the benefits of the senescent cell paradigm is that successfulremoval of senescent cells may provide the subject with a long-termtherapeutic effect. Senescent cells are essentially non-proliferative,which means that subsequent repopulation of a tissue with more senescentcells can only occur by conversion of non-senescent cells in the tissueto senescent cells—a process that takes considerably longer than simpleproliferation. As a general principle, a period of therapy with asenolytic agent that is sufficient to remove senescent cells from atarget tissue (a single dose, or a plurality of doses given, forexample, every day, semi-weekly, or weekly, given over a period of a fewdays, a week, or several months) may provide the subject with a periodof efficacy (for example, for two weeks, a month, two months, or more)during which the senolytic agent is not administered, and the subjectexperiences alleviation, reduction, or reversal of one or more adversesigns or symptoms of the condition being treated.

To treat a particular senescence-related condition with a senolyticagent according to this disclosure, the therapeutic regimen will dependon the location of the senescent cells, and the pathophysiology of thedisease.

Senescence-Related Conditions Suitable for Treatment

The Bcl inhibitors of this disclosure can be used for prevention ortreatment of various senescence-related conditions. Such conditions willtypically (although not necessarily) characterized by an overabundanceof senescent cells (such as cells expressing p16 and other senescencemarkers) in or around the site of the condition, or an overabundance ofexpression of p16 and other senescence markers, in comparison with thefrequency of such cells or the level of such expression in unaffectedtissue. Non-limiting examples of current interest include the treatmentof osteoarthritis, eye disease, and lung disease, as illustrated in thefollowing sections.

Treatment of Osteoarthritis

Any of the Bcl inhibitors listed in this disclosure can be developed fortreating osteoarthritis in accordance with this disclosure. Similarly,of the Bcl inhibitors listed in this disclosure can be developed forselectively eliminating senescent cells in or around a joint of asubject in need thereof, including but not limited to a joint affectedby osteoarthritis.

Osteoarthritis degenerative joint disease is characterized byfibrillation of the cartilage at sites of high mechanical stress, bonesclerosis, and thickening of the synovium and the joint capsule.Fibrillation is a local surface disorganization involving splitting ofthe superficial layers of the cartilage. The early splitting istangential with the cartilage surface, following the axes of thepredominant collagen bundles. Collagen within the cartilage becomesdisorganized, and proteoglycans are lost from the cartilage surface. Inthe absence of protective and lubricating effects of proteoglycans in ajoint, collagen fibers become susceptible to degradation, and mechanicaldestruction ensues. Predisposing risk factors for developingosteoarthritis include increasing age, obesity, previous joint injury,overuse of the joint, weak thigh muscles, and genetics. Symptoms ofosteoarthritis include sore or stiff joints, particularly the hips,knees, and lower back, after inactivity or overuse; stiffness afterresting that goes away after movement; and pain that is worse afteractivity or toward the end of the day.

Compounds according to this disclosure can be used to reduce or inhibitloss or erosion of proteoglycan layers in a joint, reduces inflammationin the affected joint, and promotes, stimulates, enhances, or inducesproduction of collagen, for example, type 2 collagen. The compound maycauses a reduction in the amount, or level, of inflammatory cytokines,such as IL-6, produced in a joint and inflammation is reduced. Thecompounds can be used for treating osteoarthritis and/or inducingcollagen, for example, Type 2 collagen, production in the joint of asubject. A compound also can be used for decreasing, inhibiting, orreducing production of metalloproteinase 13 (MMP-13), which degradescollagen in a joint, and for restoring proteoglycan layer or inhibitingloss and/or degradation of the proteoglycan layer. Treatment with acompound thereby may also reduce the likelihood of, inhibits, ordecreases erosion, or slows erosion of the bone. The compound may beadministered directly to an osteoarthritic joint, for example,intra-articularly, topically, transdermally, intradermally, orsubcutaneously. The compound may also restore, improve, or inhibitdeterioration of strength of a join, and reduce joint pain.

Treatment of Ophthalmic Conditions

Any of the Bcl inhibitors listed in this disclosure can be used forpreventing or treating an ophthalmic condition in a subject in needthereof by removing senescent cells in or around an eye of the subject,whereby at least one sign or symptom of the disease is decreased inseverity. Such conditions include both back-of-the-eye diseases, andfront-of-the-eye diseases. Similarly, of the Bcl inhibitors listed inthis disclosure can be developed for selectively eliminating senescentcells in or around ocular tissue in a subject in need thereof.

Diseases of the eye that can be treated according to this disclosureinclude presbyopia, macular degeneration (including wet or dry AMD),diabetic retinopathy, and glaucoma.

Macular degeneration is a neurodegenerative condition that can becharacterized as a back-of-the-eye disease. It causes the loss ofphotoreceptor cells in the central part of retina, called the macula.Macular degeneration can be dry or wet. The dry form is more common thanthe wet, with about 90% of age-related macular degeneration (AMD)patients diagnosed with the dry form. Dry AMD is associated with atrophyof the retinal pigment epithelium (RPE) layer, which causes loss ofphotoreceptor cells. With wet AMD, new blood vessels may grow beneaththe retina and leak blood and fluid. Abnormally leaky choroidalneovascularization can cause the retinal cells to die, creating blindspots in central vision. The formation of exudates, or “drusen,”underneath the Bruch's membrane of the macula is can be a physical signthat macular degeneration is emerging. Symptoms of macular degenerationinclude, for example, perceived distortion of straight lines, dark,blurry areas and color perception changes.

Another back-of-the-eye disease is diabetic retinopathy (DR). Accordingto Wikipedia, the first stage of DR is non-proliferative, and typicallyhas no substantial symptoms or signs. NPDR is detectable by fundusphotography, in which microaneurysms (microscopic blood-filled bulges inthe artery walls) can be seen. If there is reduced vision, fluoresceinangiography can be done to see the back of the eye. Narrowing or blockedretinal blood vessels can be seen clearly and this is called retinalischemia (lack of blood flow). Macular edema in which blood vessels leaktheir contents into the macular region can occur at any stage of NPDR.The symptoms of macular edema are blurred vision and darkened ordistorted images that are not the same in both eyes. Optical CoherenceTomography can show the areas of retinal thickening (due to fluidaccumulation) of macular edema. In the second stage of DR, abnormal newblood vessels (neovascularization) form at the back of the eye as partof proliferative diabetic retinopathy (PDR), which may burst and bleed(vitreous hemorrhage) and blur the vision. On funduscopic exam, aclinician will see cotton wool spots, flame hemorrhages (similar lesionsare also caused by the alpha-toxin of Clostridium novyi), and dot-blothemorrhages.

Benefits of treatment of back-of-the-eye disease with a senolytic agentof this disclosure may include inhibition or delay of adverse featuresof the condition, such as abnormal neovascularization, pathogenicangiogenesis, vaso-obliteration, intraocular bleeding, retinal damage,and vision loss. The senolytic agent may be administered in or aroundthe eye, for example, by intraocular, intravitreal, or retrobulbarinjection. Optimally, there will be a reversal in some of thepathophysiology, such as restoration of functional vasculature,functional angiogenesis, retinal regrowth or restoration, with a partialdegree of vision improvement.

Presbyopia is an age-related condition where the eye exhibits aprogressively diminished ability to focus on near objects as the speedand amplitude of accommodation of a normal eye decreases with advancingage. Loss of elasticity of the crystalline lens and loss ofcontractility of the ciliary muscles can cause presbyopia. Age-relatedchanges in the mechanical properties of the anterior lens capsule andposterior lens capsule suggest that the mechanical strength of theposterior lens capsule decreases significantly with age as a consequenceof change in the composition of the tissue. The major structuralcomponent of the lens capsule is basement membrane type IV collagen thatis organized into a three-dimensional molecular network. Adhesion of thecollagen IV, fibronectin, and lamina to the intraocular lens can inhibitcell migration and can reduce the risk of PCO.

Senolytic agents provided by this disclosure may slow thedisorganization of the type IV collagen network, decrease or inhibitepithelial cell migration and can also delay the onset of presbyopia ordecrease or slow the progressive severity of the condition. They canalso be useful for post-cataract surgery to reduce the likelihood ofoccurrence of PCO.

Glaucoma and other front-of-the-eye diseases may also be amenable totreatment with the senolytic agents provided in this disclosure.Normally, clear fluid flows into and out of the front part of the eye,known as the anterior chamber. In individuals who have open/wide-angleglaucoma, the clear fluid drains too slowly, leading to increasedpressure within the eye. If left untreated, the high pressure in the eyecan subsequently damage the optic nerve and can lead to completeblindness. The loss of peripheral vision is caused by the death ofganglion cells in the retina.

Possible benefits of therapy include a reduction in intraocularpressure, improved draining of ocular fluid through the trabecularnetwork, and an inhibition or delay of vision loss that results. Thesenolytic agent may be administered in or around the eye, for example,by intraocular or intracameral injection or in a topical formulation.The effect of therapy can be monitored by automated perimetry,gonioscopy, imaging technology, scanning laser tomography, HRT3, laserpolarimetry, GDX, ocular coherence tomography, ophthalmoscopy, andpachymeter measurements that determine central corneal thickness.

Treatment of Pulmonary Conditions

Any of the Bcl inhibitors listed in this disclosure can be developed fortreating pulmonary disease in accordance with this disclosure.Similarly, of the Bcl inhibitors listed in this disclosure can bedeveloped for selectively eliminating senescent cells in or around alung of a subject in need thereof. Pulmonary conditions that can betreated include idiopathic pulmonary fibrosis (IPF), chronic obstructivepulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, andemphysema.

COPD is a lung disease defined by persistently poor airflow resultingfrom the breakdown of lung tissue, emphysema, and the dysfunction of thesmall airways, obstructive bronchiolitis. Primary symptoms of COPDinclude shortness of breath, wheezing, chest tightness, chronic cough,and excess sputum production. Elastase from cigarette smoke-activatedneutrophils and macrophages can disintegrate the extracellular matrix ofalveolar structures, resulting in enlarged air spaces and loss ofrespiratory capacity. COPD can be caused by, for example, tobacco smoke,cigarette smoke, cigar smoke, secondhand smoke, pipe smoke, occupationalexposure, exposure to dust, smoke, fumes, and pollution, occurring overdecades thereby implicating aging as a risk factor for developing COPD.

The processes that cause lung damage include, for example, oxidativestress produced by the high concentrations of free radicals in tobaccosmoke, cytokine release due to the inflammatory response to irritants inthe airway, and impairment of anti-protease enzymes by tobacco smoke andfree radicals, allowing proteases to damage the lungs. Geneticsusceptibility can also contribute to the disease. In about 1% percentof people with COPD, the disease results from a genetic disorder thatcauses low level production of alpha-1-antitrypsin in the liver.Alpha-1-antitrypsin is normally secreted into the bloodstream to helpprotect the lungs.

Pulmonary fibrosis is a chronic and progressive lung diseasecharacterized by stiffening and scarring of the lung, which can lead torespiratory failure, lung cancer, and heart failure. Fibrosis isassociated with repair of epithelium. Fibroblasts are activated,production of extracellular matrix proteins is increased, andtransdifferentiation to contractile myofibroblasts contribute to woundcontraction. A provisional matrix plugs the injured epithelium andprovides a scaffold for epithelial cell migration, involving anepithelial-mesenchymal transition (EMT). Blood loss associated withepithelial injury induces platelet activation, production of growthfactors, and an acute inflammatory response. Normally, the epithelialbarrier heals and the inflammatory response resolves. However, infibrotic disease the fibroblast response continues, resulting inunresolved wound healing. Formation of fibroblastic foci is a feature ofthe disease, reflecting locations of ongoing fibrogenesis.

Subjects at risk of developing pulmonary fibrosis include, for example,those exposed to environmental or occupational pollutants, such asasbestosis and silicosis; those who smoke cigarettes; those who have aconnective tissue diseases such as RA, SLE, scleroderma, sarcoidosis, orWegener's granulomatosis; those who have infections; those who takecertain medications, including, for example, amiodarone, bleomycin,busufan, methotrexate, and nitrofurantoin; those subject to radiationtherapy to the chest; and those whose family member have pulmonaryfibrosis.

Other pulmonary conditions that can be treated by using a compoundaccording to this condition include emphysema, asthma, bronchiectasis,and cystic fibrosis. Pulmonary diseases can also be exacerbated bytobacco smoke, occupational exposure to dust, smoke, or fumes,infection, or pollutants that contribute to inflammation.

Symptoms of lung disease can include of shortness of breath, wheezing,chest tightness, having to clear one's throat first thing in the morningbecause of excess mucus in the lungs, a chronic cough that producessputum that can be clear, white, yellow or greenish, cyanosis, frequentrespiratory infections, lack of energy, and unintended weight loss.Symptoms of pulmonary fibrosis may include shortness of breath,particularly during exercise; dry, hacking cough; fast, shallowbreathing; gradual, unintended weight loss; fatigue; aching joints andmuscles; and clubbing of the fingers or toes.

Lung function before, during, and after treatment can be determined, forexample, by measuring expiratory reserve volume (ERV), forced vitalcapacity (FVC), forced expiratory volume (FEV), total lung capacity(TLC), vital capacity (VC), residual volume (RV), and functionalresidual capacity (FRC). Gas exchange across alveolar capillary membranecan be measured using diffusion capacity for carbon monoxide (DLCO).Exercise capacity can be measured as a proxy. Peripheral capillaryoxygen saturation (SpO₂) can also be measured: normal oxygen levels aretypically between 95% and 100%. An SpO₂ level below 90% suggests thesubject has hypoxemia. Values below 80% are considered critical andrequire intervention to maintain brain and cardiac function and avoidcardiac or respiratory arrest.

Benefits of treatment may include inhibiting progression or reversing ofany of these effects. Administration of the senolytic agent may besystemic, or local at a site in or around the lung: for example, byinhalation as an aerosol or powder, or by intubation. Optimally, theagent will improve the SpO₂ level and exercise capacity.

Treatment of Atherosclerosis

The senolytic compounds can be used for the treatment ofatherosclerosis: for example, by inhibiting formation, enlargement, orprogression of atherosclerotic plaques in a subject. The senolyticcompounds can also be used to enhance stability of atheroscleroticplaques that are present in one or more blood vessels of a subject,thereby inhibiting them from rupturing and occluding the vessels.

Atherosclerosis is characterized by patchy intimal plaques, atheromas,that encroach on the lumen of medium-sized and large arteries; theplaques contain lipids, inflammatory cells, smooth muscle cells, andconnective tissue. Atherosclerosis can affect large and medium-sizedarteries, including the coronary, carotid, and cerebral arteries, theaorta and branches thereof, and major arteries of the extremities.

Atherosclerosis may lead to an increase in artery wall thickens.Symptoms develop when growth or rupture of the plaque reduces orobstructs blood flow; and the symptoms can vary depending on whichartery is affected. Atherosclerotic plaques can be stable or unstable.Stable plaques regress, remain static, or grow slowly, sometimes overseveral decades, until they can cause stenosis or occlusion. Unstableplaques are vulnerable to spontaneous erosion, fissure, or rupture,causing acute thrombosis, occlusion, and infarction long before theycause hemodynamically significant stenosis. Clinical events can resultfrom unstable plaques, which do not appear severe on angiography; thus,plaque stabilization can be a way to reduce morbidity and mortality.Plaque rupture or erosion can lead to major cardiovascular events suchas acute coronary syndrome and stroke. Disrupted plaques can have agreater content of lipid, macrophages, and have a thinner fibrous capthan intact plaques.

Diagnosis of atherosclerosis and other cardiovascular disease can bebased on symptoms, for example, angina, chest pressure, numbness orweakness in arms or legs, difficulty speaking or slurred speech,drooping muscles in face, leg pain, high blood pressure, kidney failureand/or erectile dysfunction, medical history, and/or physicalexamination of a patient. Diagnosis can be confirmed by angiography,ultrasonography, or other imaging tests. Subjects at risk of developingcardiovascular disease include those having any one or more ofpredisposing factors, such as a family history of cardiovascular diseaseand those having other risk factors, for example, predisposing factorsincluding high blood pressure, dyslipidemia, high cholesterol, diabetes,obesity and cigarette smoking, sedentary lifestyle, and hypertension.The condition can be assessed, for example, by angiography,electrocardiography, or stress test.

Potential benefits of treatment with a senolytic agent includealleviating or halting progression of one or more signs or symptoms ofthe condition, such as the frequency of plaques, the surface area ofvessels covered by plaques, angina, and reduced exercise tolerance.

Exemplary Phospholidines

Included in the formulas presented above are the following exemplarycompounds, which can be screened and developed for the various usesdescribed as part of this disclosure.

Definitions

A “senescent cell” is generally thought to be derived from a cell typethat typically replicates, but as a result of aging or other event thatcauses a change in cell state, can no longer replicate. Depending on thecontext, senescent cells can be identified as expressing p16, or atleast one marker selected from p16, senescence-associatedβ-galactosidase, and lipofuscin; sometimes two or more of these markers,and other markers of the senescence-associated secretory profile (SASP)such as but not limited to interleukin 6, and inflammatory, angiogenicand extracellular matrix modifying proteins. Unless explicity statedotherwise, the senescent cells referred to in the claims do not includecancer cells.

A “senescence associated”, “senescence related” or “age related”disease, disorder, or condition is a physiological condition thatpresents with one or more symptoms or signs that are adverse to thesubject. The condition is “senescence associated” if it is “caused ormediated at least in part by senescent cells.” This means that at leastone component of the SASP in or around the affected tissue plays a rolein the pathophysiology of the condition such that elimination of atleast some of the senescent cells in the affected tissue results insubstantial relief or lessening of the adverse symptoms or signs, to thepatient's benefit. Senescence associated disorders that can potentiallybe treated or managed using the methods and products according to thisdisclosure include disorders referred to in this disclosure and inprevious disclosures referred to in the discussion. Unless explicitlystated otherwise, the term does not include cancer.

An inhibitor of protein function or Bcl function is a compound that to asubstantial degree prevents the target protein already expressed in atarget cell from performing an enzymatic, binding, or regulatoryfunction that the protein or Bcl family member normally performs in thetarget cell. This results in elimination of the target cell or renderingthe cell more susceptible to the toxicity of another compound or event.A compound qualifies as a “Bcl inhibitor” or a compound that “inhibitsBcl activity” in this disclosure if it has an IC₅₀ when tested in anassay according to Example 1 below that is less than 1,000 nM (1.0 μM).Activity that is less than 100 nM or 10 nM, or between 100 nM and 1 nMis often preferred, depending on the context.

The term “Bcl” or “Bcl protein” refers to the family of Bcl proteins,exemplified by Bcl-2, Bcl-xL, and Bcl-w. A Bcl inhibitor of thisdisclosure will be able to inhibit at least one of Bcl-2, Bcl-xL, andBcl-w. Typically but not necessarily, an inhibitor of one of these Bclproteins will to some extent inhibit the other two. The compoundsprovided in this disclosure can be tested for activity of any Bcl familymembers, to identify compounds that have inhibitory activity and arepotentially specific for Bcl-2, Bcl-xL, or Bcl-w. Such an inhibitor willhave an IC₅₀ for a target Bcl from this list that is at least 10-foldbetter than its IC₅₀ for the other two Bcl family members on the list.

A compound, composition or agent is typically referred to as “senolytic”if it eliminates senescent cells, in preference replicative cells of thesame tissue type, or quiescent cells lacking SASP markers. Alternativelyor in addition, a compound or combination may effectively be used if itdecreases the release of pathological soluble factors or mediators aspart of the senescence associated secretory phenotype that play a rolein the initial presentation or ongoing pathology of a condition, orinhibit its resolution. In this respect, the term “senolytic” refers tofunctional inhibition, such that compounds that work primarily byinhibiting rather than eliminating senescent cells (senescent cellinhibitors) can be used in a similar fashion with ensuing benefits.Model senolytic compositions and agents in this disclosure have an EC₅₀when tested in an assay according to Example 2 below that is less than 1μM. Activity that is less than 0.1 μM, or between 1 μM and 0.1 μM may bepreferred. The selectivity index (SI) (EC₅₀ of senescent cells comparedwith non-senescent cells of the same tissue type) may be better than 1,2, 5, or 10, depending on the context.

Selective removal or “elimination” of senescent cells from a mixed cellpopulation or tissue doesn't require that all cells bearing a senescencephenotype be removed: only that the proportion of senescent cellsinitially in the tissue that remain after treatment is substantiallyhigher than the proportion of non-senescent cells initially in thetissue that remain after the treatment.

Successful “treatment” of a condition according to this disclosure mayhave any effect that is beneficial to the subject being treated. Thisincludes decreasing severity, duration, or progression of a condition,or of any adverse signs or symptoms resulting therefrom. Treatment mayalso be unsuccessful, resulting in no improvement in typical signs andsymptoms of the condition. A concurrent objective of therapy is tominimize adverse effects on the target tissue or elsewhere in thetreated subject. In some circumstances, senolytic agents can also beused to prevent or inhibit presentation of a condition for which asubject is susceptible, for example, because of an inheritedsusceptibility of because of medical history.

A “therapeutically effective amount” is an amount of a compound of thepresent disclosure that (i) treats the particular disease, condition, ordisorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein, (iv) prevents ordelays progression of the particular disease, condition or disorder, or(v) at least partially reverses damage caused by the condition prior totreatment.

A “phosphorylated” form of a compound is a compound which bears one ormore phosphate groups covalently bound to the core structure through anoxygen atom, which was typically but not necessarily present on themolecule before phosphorylation. For example, one or more —OH or —COOHgroups may have been substituted in place of the hydrogen with aphosphate group which is either —OPO₃H₂ or —CnPO₃H₂ (where n is 1 to 4).In some phosphorylated forms, the phosphate group may be removed in vivo(for example, by enzymolysis), in which case the phosphorylated form maybe a 0 of the non-phosphorylated form. A non-phosphorylated form has nosuch phosphate group. A dephosphorylated form is a derivative of aphosphorylated molecule after at least one phosphate group has beenremoved.

“Small molecule” Bcl inhibitors according to this disclosure havemolecular weights less than 20,000 daltons, and are often less than10,000, 5,000, or 2,000 daltons. Small molecule inhibitors are notantibody molecules or oligonucleotides, and typically have no more thanfive hydrogen bond donors (the total number of nitrogen—hydrogen andoxygen—hydrogen bonds), and no more than 10 hydrogen bond acceptors (allnitrogen or oxygen atoms).

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. Thetransformation can be an enzymatic transformation. Sometimes, thetransformation is a cyclization transformation, or a combination of anenzymatic transformation and a cyclization transformation. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the active agent.

“Promoiety” refers to a form of protecting group that when used to maska functional group within an active agent converts the active agent intoa prodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.Exemplary promoiety groups include acyl groups capable of forming anester or thioester group with a hydroxyl or thiol functional group of acompound, and substituted alkyl groups capable of forming an ether orthioether group with a hydroxyl or thiol functional group of a compound,which groups can be cleaved in vivo as described above.

Unless otherwise stated or required, each of the compound structuresreferred to in the disclosure include conjugate acids and bases havingthe same structure, crystalline and amorphous forms of those compounds,pharmaceutically acceptable salts, and prodrugs. This includes, forexample, tautomers, polymorphs, solvates, hydrates, unsolvatedpolymorphs (including anhydrates).

Unelss otherwise stated or implied, the term “substituted” when used tomodify a specified group or radical means that one or more hydrogenatoms of the specified group or radical are each independently replacedwith the same or different substituent groups which is not hydrogen.Unless indicated otherwise, the nomenclature of substituents is arrivedat by naming the terminal portion of the functionality followed by theadjacent functionality toward the point of attachment. For example, thesubstituent “arylalkyloxycarbonyl” refers to the group(aryl)-(alkyl)-O—C(O)—.

A “linker” is a moiety that covalently connects two or more chemicalstrucutres, and has a backbone of 100 atoms or less in length betweenthe two strucdtures. The linker may be cleavable or non-cleavable. Thelinker typically has a backbone of between 1 and 20 or between 1 and 100atoms in length, in linear or branched form. The bonds between backboneatoms may be saturated or unsaturated. The linker backbone may include acyclic group, for example, an optionally substituted aryl, heteroaryl,heterocycle or cycloalkyl group.

For any Bcl inhibitor compound of this disclosure having one or morechiral centers, if an absolute stereochemistry is not expresslyindicated, then each chiral center may independently be ofR-configuration or S-configuration or a racemic mixture thereof. Thecompound may be any stereoisomer of the structure shown, either as analternative form or as a mixture, unless a particular stereoisomer isexplicity referred to.

Except where otherwise stated or required, other terms used in thespecification have their ordinary meaning.

INCORPORATION BY REFERENCE

For all purposes in the United States and in other jurisdictions whereeffective, each and every publication and patent document cited in thisdisclosure is hereby incorporated herein by reference in its entiretyfor all purposes to the same extent as if each such publication ordocument was specifically and individually indicated to be incorporatedherein by reference.

US 2016/0339019 A1 (Laberge et al.) and US 20170266211 A1 (David et al.)are hereby incorporated herein for all purposes, including but notlimited to the identification, formulation, and use of compounds capableof eliminating or reducing the activity of senescent cells and treatingparticular senescence-related conditions, including but not limited tothose referred to in this disclosure. Pre-grant patent publications US2018/0000816 A1 (David et al.) and EP 3441069 A1 (Hopkins et al.) arehereby incorporated herein for all purposes, including but not limitedto the identification, formulation, and use of compounds capable ofeliminating or reducing the activity of senescent cells and treatingvarious ophthalmic conditions.

EXAMPLES Example 1: Measuring Bcl Inhibition

The ability of candidate compounds to inhibit Bcl-2 and Bcl-xL activitycan be measured on the molecular level by direct binding. This assayuses a homogenous assay technology based on oxygen channeling that ismarketed by PerkinElmer Inc., Waltham, Mass.: see Eglin et al., CurrentChemical Genomics, 2008, 1, 2-10. The test compound is combined with thetarget Bcl protein and a peptide representing the corresponding cognateligand, labeled with biotin. The mixture is then combined withstreptavidin bearing luminescent donor beads and luminescent acceptorbeads, which proportionally reduces luminescence if the compound hasinhibited the peptide from binding to the Bcl protein.

Bcl-2, Bcl-xL and Bcl-w are available from Sigma-Aldrich Co., St. Louis,Mo. Biotinylated BIM peptide (ligand for Bcl-2) and BAD peptide (ligandfor Bcl-xL) are described in US 2016/0038503 A1. AlphaScreen®Streptavidin donor beads and Anti-6XHis AlphaLISA® acceptor beads areavailable from PerkinElmer.

To conduct the assay, a 1:4 dilution series of the compound is preparedin DMSO, and then diluted 1:100 in assay buffer. In a 96-well PCR plate,the following are combined in order: 10 μL peptide (120 nM BIM or 60 nMBIM), 10 μL test compound, and 10 μL Bcl protein (0.8 nM Bcl-2/W or 0.4nM Bcl-XL). The assay plate is incubated in the dark at room temperaturefor 24 h. The next day, donor beads and acceptor beads are combined, and5 μL is added to each well. After incubating in the dark for 30 minute,luminescence is measured using a plate reader, and the affinity ordegree of inhibition by each test compound is determined.

Example 2: Measuring Senolytic Activity in Fibroblasts

Human fibroblast IMR90 cells can be obtained from the American TypeCulture Collection (ATCC®) with the designation CCL-186. The cells aremaintained at <75% confluency in DMEM containing FBS and Pen/Strep in anatmosphere of 3% O₂, 10% CO₂, and ˜95% humidity. The cells are dividedinto groups: irradiated cells (cultured for 14 days after irradiationprior to use) and quiescent cells (cultured at high density for four dayprior to use).

On day 0, the irradiated cells are prepared as follows. IMR90 cells arewashed, placed in T175 flasks at a density of 50,000 cells per mL, andirradiated at 10-15 Gy. Following irradiation, the cells are plated at100 μL in 96-well plates. On days 1, 3, 6, 10, and 13, the medium ineach well is aspirated and replaced with fresh medium.

On day 10, the quiescent healthy cells are prepared as follows. IMR90cells are washed, combined with 3 mL of TrypLE trypsin-containingreagent (Thermofisher Scientific, Waltham, Mass.) and cultured for 5 minuntil the cells have rounded up and begin to detach from the plate.Cells are dispersed, counted, and prepared in medium at a concentrationof 50,000 cells per mL. 100 μL of the cells is plated in each well of a96-well plate. Medium is changed on day 13.

On day 14, test inhibitor compounds are combined with the cells asfollows. A DMSO dilution series of each test compound is prepared at 200times the final desired concentration in a 96-well PCR plate.Immediately before use, the DMSO stocks are diluted 1:200 into prewarmedcomplete medium. Medium is aspirated from the cells in each well, and100 μL/well of the compound containing medium is added.

Candidate senolytic agents for testing are cultured with the cells for 6days, replacing the culture medium with fresh medium and the samecompound concentration on day 17. Bcl 2 inhibitors are cultured with thecells for 3 days. The assay system uses the properties of a thermostableluciferase to enable reaction conditions that generate a stableluminescent signal while simultaneously inhibiting endogenous ATPasereleased during cell lysis. At the end of the culture period, 100 μL ofCellTiter-Glo® reagent (Promega Corp., Madison, Wis.) is added to eachof the wells. The cell plates are placed for 30 seconds on an orbitalshaker, and luminescence is measured.

Example 3: Measuring Senolytic Activity in HUVEC Cells and OtherSenescent Cells

Human umbilical vein (HUVEC) cells from a single lot were expanded inVascular Cell Basal Media supplemented with the Endothelial Cell GrowthKit™-VEGF from ATCC to approximately eight population doublings thencryopreserved. Nine days prior to the start of the assay, cells for thesenescent population were thawed and seeded at approximately 27,000/cm₂.All cells were cultured in humidified incubators with 5% CO₂ and 3% O₂and media was changed every 48 hr. Two days after seeding, the cellswere irradiated, delivering 12 Gy radiation from an X-ray source. Threedays prior to the start of the assay, cells for the non-senescentpopulations are thawed and seeded as for the senescent population. Oneday prior to the assay, all cells were trypsinized and seeded into384-well plates, 5,000/well senescent cells and 10,000/wellnon-senescent in separate plates in a final volume of 55 μL/well. Ineach plate, the central 308 wells contained cells and the outerperimeter of wells was filled with 70 μL/well deionized water.

On the day of the assay, compounds were diluted from 10 mM stocks intomedia to provide the highest concentration working stock, aliquots ofwhich were then further diluted in media to provide the remaining twoworking stocks. To initiate the assay, 5 μL of the working stock wasadded to the cell plates. The final test concentrations were 20, 2, and0.2 μM. In each plate, 100 test compounds were assayed in triplicate ata single concentration along with a three wells of a positive controland five no treatment (DMSO) controls. Following compound addition, theplates are returned to the incubators for three days.

Cell survival was assessed indirectly by measuring total ATPconcentration using CellTiter-Glo™ reagent (Promega). The resultantluminescence was quantitated with an EnSpire™ plate reader (PerkinElmer). The relative cell viability for each concentration of a compoundwas calculated as a percentage relative to the no-treatment controls forthe same plate.

For follow-up dose responses of potential lead compounds, 384-wellplates of senescent and non-senescent cells were prepared as describedabove. Compounds were prepared as 10-point 1:3 dilution series in DMSO,then diluted to 12× in media. Five microliters of this working stock wasthen added to the cell plates. After three days of incubation, cellsurvival relative to DMSO control was calculated as described above. Allmeasurements were performed in quadruplicate.

Other cell lines and primary cell cultures may be used as an alternativeto IMR90 fibroblasts or HUVEC cells that align with the intended targettissue in vivo. An example is the use of cultured human retinalmicrovascular endothelial cells (HRMEC) for screening compounds intendedfor treatment of eye disease. The cells are cultured according to knownprotocols for the chosen cell line, and irradiated in a similar fashionto render them senescent.

Example 4: Efficacy of Senolytic Agents in an Osteoarthritis Model

This example illustrates the testing of an MDM2 inhibitor in a mousemodel for treatment of osteoarthritis. It can be adapted mutatismutandis to test and develop Bcl inhibitors for use in clinical therapy.

The model was implemented as follows. C₅₇BL/6J mice underwent surgery tocut the anterior cruciate ligament of one rear limb to induceosteoarthritis in the joint of that limb. During week 3 and week 4post-surgery, the mice were treated with 5.8 μg of Nutlin-3A (n=7) peroperated knee by intra-articular injection, q.o.d. for 2 weeks. At theend of 4 weeks post-surgery, joints of the mice were monitored forpresence of senescent cells, assessed for function, monitored formarkers of inflammation, and underwent histological assessment.

Two control groups of mice were included in the studies performed: onegroup comprising C57BL/6J or 3MR mice that had undergone a sham surgery(n=3) (i.e., surgical procedures followed except for cutting the ACL)and intra-articular injections of vehicle parallel to the GCV(ganciclovir) treated group; and one group comprising C57BL/6J or 3MRmice that had undergone an ACL surgery and received intra-articularinjections of vehicle (n=5) parallel to the GCV-treated group. RNA fromthe operated joints of mice from the Nutlin-3A treated mice was analyzedfor expression of SASP factors (mmp3, IL-6) and senescence markers(p16). qRT-PCR was performed to detect mRNA levels.

FIGS. 5A, 5B, and 5C show expression of p16, IL-6, and MMP13 in thetissue, respectively. The OA inducing surgery was associated withincreased expression of these markers. Treatment with Nutlin-3A reducedthe expression back to below the level of the controls. Treatment withNutlin-3A cleared senescent cells from the joint.

Function of the limbs was assessed 4 weeks post-surgery by a weightbearing test to determine which leg the mice favored. The mice wereallowed to acclimate to the chamber on at least three occasions prior totaking measurements. Mice were maneuvered inside the chamber to standwith one hind paw on each scale. The weight that was placed on each hindlimb was measured over a three second period. At least three separatemeasurements were made for each animal at each time point. The resultswere expressed as the percentage of the weight placed on the operatedlimb versus the contralateral unoperated limb.

FIG. 6A shows the results of the functional study. Untreated mice thatunderwent osteoarthritis inducing surgery favored the unoperated hindlimb over the operated hind limb (Δ). However, clearing senescent cellswith Nutlin-3A abrogated this effect in mice that have undergone surgery(∇).

FIGS. 6B, 6C, and 6D show histopathology of joint tissue from theseexperiments. Osteoarthritis induced by ACL surgery caused theproteoglycan layer was destroyed. Clearing of senescent cells usingNutlin-3A completely abrogated this effect.

Example 5: Efficacy of Senolytic Agents in Models for DiabeticRetinopathy

This example illustrates the testing of a Bcl inhibitor in a mouse modelfor treatment of a back-of-the eye disease, specifically diabeticretinopathy. It can be adapted mutatis mutandis to test senolytic agentsfor use in clinical therapy.

The efficacy of model compound UBX1967 (a Bcl-xL inhibitor) was studiedin the mouse oxygen-induced retinopathy (OIR) model (Scott andFruttiger, Eye (2010) 24, 416-421, Oubaha et al, 2016). 057Bl/6 mousepups and their CD1 foster mothers were exposed to a high oxygenenvironment (75% O₂) from postnatal day 7 (P7) to P12. At P12, animalswere injected intravitreally with 1 μl test compound (200, 20, or 2 uM)formulated in 1% DMSO, 10% Tween-80, 20% PEG-400, and returned to roomair until P17. Eyes were enucleated at P17 and retinas dissected foreither vascular staining or qRT-PCR. To determine avascular orneovascular area, retinas were flat-mounted, and stained with isolectinB4 (IB4) diluted 1:100 in 1 mM CaCl₂). For quantitative measurement ofsenescence markers (e.g., Cdkn2a, Cdknla, 116, Vegfa), qPCR wasperformed. RNA was isolated and cDNA was generated byreverse-transcription, which was used for qRT-PCR of the selectedtranscripts.

FIGS. 7A and 7B show that intravitreal ITT) administration UBX1967resulted in statistically significant improvement in the degree ofneovascularization and vaso-obliteration at all dose levels.

The efficacy of UBX1967 was also studied in the streptozotocin (STZ)model. C₅₇BL/6J mice of 6- to 7-week were weighted and their baselineglycemia was measured (Accu-Chek™, Roche). Mice were injectedintraperitoneally with STZ (Sigma-Alderich, St. Louis, Mo.) for 5consecutive days at 55 mg/Kg. Age-matched controls were injected withbuffer only. Glycemia was measured again a week after the last STZinjection and mice were considered diabetic if their non-fasted glycemiawas higher than 17 mM (300 mg/L). STZ treated diabetic C57BL/6J micewere intravitreally injected with 1 μl of UBX1967 (2 μM or 20 μM,formulated as a suspension in 0.015% polysorbate-80, 0.2% SodiumPhosphate, 0.75% Sodium Chloride, pH 7.2) at 8 and 9 weeks after STZadministration. Retinal Evans blue permeation assay was performed at 10weeks after STZ treatment.

FIGS. 7C and 7D show results for this protocol. Retinal and choroidalvascular leakage after intravitreal (IVT) administration UBX1967improved in vascular permeability at both dose levels.

Other models of retinal ganglion cell damage can be used in testing thatare relevant to glaucoma, where increased intraocular pressure (IOP) isthought to cause retinal ganglion cell loss and optic nerve damage. Inpreclinical species, increased anterior chamber pressure can result inretinal neuron loss as reported in several established models, includingthe magnetic microbead occlusion (Ito et al., Vis Exp. 2016 (109):53731) and other glaucoma models (Almasieh and Levin, Annu Rev Vis Sci.2017). Additionally, ischemia-reperfusion has been demonstrated to causeretinal injury which may result in cellular senescence. Presence ofretinal senescence in such models can be used to monitor the impact ofsenolysis after intravitreal injection of test compounds.

Example 6: Efficacy of Senolytic Agents in a Pulmonary Disease Model

This example illustrates the testing of inhibitors in a mouse model fortreatment of lung disease: specifically, a model for idiopathicpulmonary fibrosis (IPF). It can be adapted mutatis mutandis to test anddevelop Bcl inhibitors for use in clinical therapy.

As a model for chronic obstructive pulmonary disease (COPD), mice wereexposed to cigarette smoke.

The effect of a senolytic agent on the mice exposed to smoke is assessedby senescent cell clearance, lung function, and histopathology.

The mice used in this study include the 3MR strain, described in US2017/0027139 A1 and in Demaria et al., Dev Cell. 2014 Dec. 22; 31(6):722-733. The 3MR mouse has a transgene encoding thymidine kinase thatconverts the prodrug ganciclovir (GCV) to a compound that is lethal tocells. The enzyme in the transgene is placed under control of the p16promoter, which causes it to be specifically expressed in senescentcells. Treatment of the mice with GCV eliminates senescent cells.

Other mice used in this study include the INK-ATTAC strain, described inUS 2015/0296755 A1 and in Baker et al., Nature 2011 Nov. 2;479(7372):232-236. The INK-ATTAC mouse has a transgene encodingswitchable caspase 8 under control of the p16 promoter. The caspase 8can be activated by treating the mice with the switch compound AP20187,whereupon the caspase 8 directly induces apoptosis in senescent cells,eliminating them from the mouse.

To conduct the experiment, six-week-old 3MR (n=35) or INK-ATTAC (n=35)mice were chronically exposed to cigarette smoke generated from a TeagueTE-10 system, an automatically-controlled cigarette smoking machine thatproduces a combination of side-stream and mainstream cigarette smoke ina chamber, which is transported to a collecting and mixing chamber wherevarying amounts of air is mixed with the smoke mixture. The COPDprotocol was adapted from the COPD core facility at Johns HopkinsUniversity (Rangasamy et al., 2004, J. Clin. Invest. 114:1248-1259; Yaoet al., 2012, J. Clin. Invest. 122:2032-2045).

Mice received a total of 6 hours of cigarette smoke exposure per day, 5days a week for 6 months. Each lighted cigarette (3R4F researchcigarettes containing 10.9 mg of total particulate matter (TPM), 9.4 mgof tar, and 0.726 mg of nicotine, and 11.9 mg carbon monoxide percigarette [University of Kentucky, Lexington, Ky.]) was puffed for 2seconds and once every minute for a total of 8 puffs, with the flow rateof 1.05 L/min, to provide a standard puff of 35 cm3. The smoke machinewas adjusted to produce a mixture of side stream smoke (89%) andmainstream smoke (11%) by smoldering 2 cigarettes at one time. The smokechamber atmosphere was monitored for total suspended particulates(80-120 mg/m3) and carbon monoxide (350 ppm).

Beginning at day 7, (10) INK-ATTAC and (10) 3MR mice were treated withAP20187 (3× per week) or ganciclovir (5 consecutive days of treatmentfollowed by 16 days off drug, repeated until the end of the experiment),respectively. An equal number of mice received the correspondingvehicle. The remaining 30 mice (15 INK-ATTAC and 15 3MR) were evenlysplit with 5 of each genetically modified strain placed into threedifferent treatment groups. One group (n=10) received Nutlin-3A (25mg/kg dissolved in 10% DMSO/3% Tween-20™ in PBS, treated 14 daysconsecutively followed by 14 days off drug, repeated until the end ofthe experiment). One group (n=10) received ABT-263 (Navitoclax) (100mg/kg dissolved in 15% DMSO/5% Tween-20, treated 7 days consecutivelyfollowed by 14 days off drug, repeated until the end of the experiment),and the last group (n=10) received only the vehicle used for ABT-263(15% DMSO/5% Tween-20), following the same treatment regimen as ABT-263.An additional 70 animals that did not receive exposure to cigarettesmoke were used as controls for the experiment.

After two months of cigarette smoke (CS) exposure, lung function wasassessed by monitoring oxygen saturation using the MouseSTATPhysioSuite™ pulse oximeter (Kent Scientific). Animals were anesthetizedwith isoflurane (1.5%) and the toe clip was applied. Mice were monitoredfor 30 seconds and the average peripheral capillary oxygen saturation(SpO₂) measurement over this duration was calculated.

FIG. 8 shows the results. Clearance of senescent cells via AP2018,ganciclovir, ABT-263 (Navitoclax), or Nutlin-3A resulted instatistically significant increases in SpO₂ levels in mice after twomonths of cigarette smoke exposure, compared with untreated controls.

Example 7: Efficacy of Senolytic Agents in Atherosclerosis whenAdministered Systemically

This example illustrates the testing of an MDM2 inhibitor in a mousemodel for treatment of atherosclerosis. The test compounds areadministered systemically rather than locally. The model is done in anLDLR−/− strain of mice, which are deficient in the receptor forlow-density lipoprotein. The experiments described here can be adaptedmutatis mutandis to test and develop other types of inhibitors for usein clinical therapy.

Two groups of LDLR−/− mice (10 weeks) are fed a high fat diet (HFD)(Harlan Teklad TD.88137) having 42% calories from fat, beginning at Week0 and throughout the study. Two groups of LDLR−/− mice (10 weeks) arefed normal chow (−HFD). From weeks 0-2, one group of HFD mice and −HFDmice are treated with Nutlin-3A (25 mg/kg, intraperitoneally). Onetreatment cycle is 14 days treatment, 14 days off. Vehicle isadministered to one group of HFD mice and one group of −HFD mice. Atweek 4 (timepoint 1), one group of mice are sacrificed and to assesspresence of senescent cells in the plaques. For the some of theremaining mice, Nutlin-3A and vehicle administration is repeated fromweeks 4-6. At week 8 (timepoint 2), the mice are sacrificed and toassess presence of senescent cells in the plaques. The remaining miceare treated with Nutlin-3A or vehicle from weeks 8-10. At week 12(timepoint 3), the mice are sacrificed and to assess the level of plaqueand the number of senescent cells in the plaques.

Plasma lipid levels were measured in LDLR−/− mice fed a HFD and treatedwith Nutlin-3A or vehicle at timepoint 1 as compared with mice fed a−HFD (n=3 per group). Plasma was collected mid-afternoon and analyzedfor circulating lipids and lipoproteins.

At the end of timepoint 1, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle were sacrificed (n=3, all groups), and the aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR−/− mice on a normaldiet. The data show that clearance of senescent cells with Nutlin-3A inLDLR−/− mice fed a HFD reduced expression of several SASP factors andsenescent cell markers, MMP3, MMP13, PAI1, p21, IGFBP2, IL-1A, and IL-1B after one treatment cycle.

At the end of timepoint 2, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR−/− mice on a normaldiet. The data show expression of some SASP factors and senescent cellmarkers in the aortic arch within HFD mice. Clearance of senescent cellswith multiple treatment cycles of Nutlin-3A in LDLR−/− mice fed a HFDreduced expression of most markers.

At the end of timepoint 3, LDLR−/− mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aortaswere dissected and stained with Sudan IV to detect the presence oflipid. Body composition of the mice was analyzed by MRI, and circulatingblood cells were counted by Hemavet™.

FIG. 9 shows the results. Treatment with Nutlin-3A reduced the surfacearea covered by plaques in the descending aorta by about 45%. Theplatelet and lymphocyte counts were equivalent between the Nutlin-3A andvehicle treated mice. Treatment with Nutlin-3A also decreased mass andbody fat composition in mice fed the high fat diet.

Example 8: Measuring Cytotoxicity for Cancer Cells In Vitro and In Vivo

The cellular activity of compounds can be evaluated in the interleukin-3(IL-3)-dependent prolymphocytic FL5.12 murine cell line. Withdrawal ofIL-3 induces FL5.12 apoptosis, by up-regulation of the proapoptoticfactors Bim and Puma. Overexpression of Bcl-2 (FL5.12-Bcl-2) or Bcl-xL(FL5.12-Bcl-xL) protects against the effects of IL-3 withdrawal bysequestration of Bim and Puma. Compounds reverse the protection affordedby overexpression of Bcl-2 or Bcl-xL. Compounds are ineffective ineliciting cell death in the presence of IL-3 where FL5.12 cells are notsubject to proapoptotic stimuli. The ability of compounds to killFL5.12-Bcl-2 or FL5.12-Bcl-xL cells under IL-3 withdrawal can beattenuated in the presence of the caspase inhibitor ZVAD, indicatingthat cell killing is caspase dependent.

Co-immunoprecipitation studies can be done to determine if BH3 mimeticinduced cytotoxicity can be attributed to the disruption ofintracellular Bcl-2 family protein-protein interactions. Compoundsinduce a dose-dependent decrease in Bim:Bcl-xL interactions inFL5.12-Bcl-xL cells. Similar results are also observed for thedisruption of Bim:Bcl-2 complexes in FL5.12-Bcl-2 cells indicating thatcompounds restore IL-3-dependent cell death by attenuating the abilityof Bcl-xL and Bcl-2 to sequester proapoptotic factors such as Bim.

Testing of the ability of compounds listed in this disclosure tospecifically kill cancer cells can be tested in similar assays usingother established cell lines. These include HeLa cells, OVCAR-3, LNCaP,and any of the Authenticated Cancer Cell Lines available from MilliporeSigma, Burlington Mass., U.S.A. Compounds specifically kill cancer cellsif they are lethal to the cells at a concentration that is at least5-fold lower, and preferably 25- or 100-fold lower than a non-cancerouscell of the same tissue type. The control cell has morphologic featuresand cell surface markers similar to the cancer cell line being tested,but without signs of cancer.

In vivo, compounds are evaluated in flank xenograft models establishedfrom sensitive SCLC (H889) and hematologic (RS4;11) cell lines, or usingother tumor-forming cancer cell lines, according to what type of canceris of particular interest to the user. When dosed orally orintravenously, compounds induce rapid and complete tumor responses (CR)that are durable for several weeks after the end of treatment in allanimals bearing H889 (SCLC) or RS4;11 (ALL) tumors. Similar treatment ofmice bearing H146 SCLC tumors can induce rapid regressions in theanimals.

Example 9: Synthesis of Phospholidine Compounds

FIGS. 1A, 1B, and 1C show a general synthetic scheme that can be used toprepare acyl phosphonamidates

FIGS. 2A, 2B, and 2C show a method that was used to synthesize ethylN-(4-(4-((4′-chloro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)-P-(4-(((R)-4-morpholino-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)phosphonamidate.The steps were as follows:

Synthesis of dimethyl (4-fluoro-3-nitrophenyl)phosphonate

To a solution of 4-iodo-2-nitro-fluorobenzene (4.55 g, 17.0 mmol) in drydioxane (40 mL) was added dimethyl phosphite (1.88 g, 17 mmol) andtriethylamine (2.59 mL, 18.7 mmol). Xantphos (492 mg, 0.85 mmol) andPd₂(dba)₃ (394 mg, 0.43 mmol) were then added under nitrogen atmosphereand the mixture was stirred at room temperature for 2.5 h. The mixturewas diluted in water (100 mL) and 1 M aqueous hydrochloric acid (20 mL)and was extracted with ethyl acetate (2×100 mL). The combined organicextracts were washed with brine (40 mL), dried over MgSO₄, filtered andconcentrated under vacuum. The crude residue was purified by silicaflash chromatography (Reveleris, 50-100% ethyl acetate in hexanes) togive dimethyl (4-fluoro-3-nitrophenyl)phosphonate as a pale yellow solid(3.09 g, 73% yield). LCMS (ESI) M+H=250.1

Synthesis of tert-butyl(R)-(4-(dimethylamino)-1-(phenylthio)butan-2-yl)carbamate

To a solution of tert-butylN-[(2R)-4-oxo-1-(phenylsulfanyl)butan-2-yl]carbamate (1 g, 3.39 mmol) ina mixture of THF (10 mL) and dimethylamine (0.95 mL of a 33% solution inethanol, 5.1 mmol) was added sodium triacetoxyborohydride (1.44 g, 6.78mmol). The mixture was stirred at room temperature for 30 min. Themixture was diluted in saturated aqueous ammonium chloride (10 mL),water (5 mL), brine (50 mL) and ethyl acetate (50 mL). The layers wereseparated and the aqueous phase pH was adjusted to ˜10 by addition ofsaturated aqueous sodium carbonate and was extracted with ethyl acetate(2×60 mL). The combined organic extracts were washed with brine (30 mL),dried over MgSO₄, filtered and concentrated under vacuum. The cruderesidue was purified by silica flash chromatography (Reveleris, 20 gsilica cartridge, 0-30% methanol in DCM) to give tert-butyl(R)-(4-(dimethylamino)-1-(phenylthio)butan-2-yl)carbamate as a colorlessoil (921 mg, 84% yield). LCMS (ESI) M+H=325.2

Synthesis of ethyl-2-(3-bromo-5-fluorobenzylidene)-3-oxobutanoate (mixof E and Z)

To a solution of 3-bromo-5-fluorobenzaldehyde (40.8 g, 201 mmol) intoluene (80 mL) and glacial acetic acid (4 mL) were added ethylacetoacetate (28.5 mL, 225 mmol) and piperidine (1.2 mL). The mixturewas heated to reflux for 2.5 h and the liberated water was collectedwith a Dean-Stark apparatus. The mixture was diluted in a mixture oftoluene (100 mL), EtOAc (200 mL) and hexanes (100 mL). The resultingsolution was washed with 1 M aqueous HCl (100 mL), water (100 mL),saturated aqueous NaHCO₃ (50 mL) and brine (50 mL). The organic layerwas dried over MgSO₄, filtered and concentrated under vacuum. The cruderesidue was treated with hot hexanes (100 mL), cooled to roomtemperature and further cooled in an ice/acetone bath. The solidmaterial was collected by filtration, washed with ice cold hexanes anddried under vacuum to giveethyl-2-(3-bromo-5-fluorobenzylidene)-3-oxobutanoate as a yellow solid(38.18 g, 60.3% yield). The filtrate was concentrated to 80 mL and theseparated material was collected by filtration and purified by silicaflash chromatography (Reveleris, 40 g cartridge, 0-100% DCM in hexanes)to give additional desired product as a yellow solid (8.8 g, 13.9%yield). LCMS (ESI) [M+H]+=315.0/317.0 [M+Na]+=337.0/339.0

Synthesis of ethyl2-acetyl-3-(3-bromo-5-fluorophenyl)-4-(4-chlorophenyl)-4-oxobutanoate

To a solution of ethyl-2-(3-bromo-5-fluorobenzylidene)-3-oxobutanoate(46.8 g, 148.5 mmol) and 4-chlorobenzaldehyde (21.4 g, 152.2 mmol) inethanol (240 mL) was added triethylamine (31 mL, 222 mmol). Nitrogen gaswas bubbled through the mixture for 5 min and3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride (6 g, 22 mmol)was then added. The mixture was heated to 70° C. for 2 h under anitrogen atmosphere. The solvent was removed under vacuum and theresidual crude was dissolved in EtOAc (700 mL) and washed with 1 Maqueous HCl (130 mL), water (2×150 mL) and brine (2×20 mL). The organicextract was dried over MgSO₄, filtered and concentrated under vacuum togive ethyl2-acetyl-3-(3-bromo-5-fluorophenyl)-4-(4-chlorophenyl)-4-oxobutanoate asan orange gum (73.1 g) which was used without purification. LCMS (ESI)[M+Na]+=477.0/479.0

Synthesis of ethyl4-(3-bromo-5-fluorophenyl)-5-(4-chlorophenyl)-1-isopropyl-2-methyl-pyrrole-3-carboxylate

To a solution of ethyl2-acetyl-3-(3-bromo-5-fluorophenyl)-4-(4-chlorophenyl)-4-oxobutanoate(73.1 g) in glacial acetic acid (140 mL) was added isopropylamine (85mL) dropwise over 15 min whilst nitrogen gas was bubbled through themixture. The mixture was allowed to stir at room temperature for 10 minthen heated to 130° C. for 2 h under a nitrogen atmosphere. The mixturewas diluted with EtOAc (600 mL) and treated with water (500 mL) andbrine (50 mL). 1 M aqueous HCl was added until reaching pH ˜1. Theorganic layer was separated, washed with water (4×500 mL), saturatedaqueous NaHCO₃ (100 mL) and brine (50 mL), dried over MgSO₄, filteredand concentrated under vacuum. The crude residue was treated with hotmethanol (250 mL) and the resulting solution was allowed to cool to roomtemperature then further cooled in an ice-acetone bath. The precipitatedsolid was collected by filtration, washed with cold methanol and driedunder vacuum to give ethyl4-(3-bromo-5-fluorophenyl)-5-(4-chlorophenyl)-1-isopropyl-2-methyl-pyrrole-3-carboxylateas a pale yellow solid (44.4 g, 62% yield over 2 steps). LCMS (ESI)single major peak, poor ionization

Synthesis of3-(3-bromo-5-fluorophenyl)-2-(4-chlorophenyl)-5-methyl-1-(propan-2-yl)-1H-pyrrole

To trifluoroacetic acid (190 mL) was added ethyl4-(3-bromo-5-fluorophenyl)-5-(4-chlorophenyl)-1-isopropyl-2-methyl-pyrrole-3-carboxylate(44.3 g, 92.5 mmol). The mixture was heated to reflux for 2 h. Themixture was poured into ice-water (800 mL) and was extracted with EtOAc(800 mL). The combined organic extracts were washed with water (4×500mL) and saturated aqueous Na₂CO₃ until reaching pH >9. The organic layerwas then washed with brine (50 mL), dried over MgSO₄, filtered andconcentrated under vacuum to give3-(3-bromo-5-fluorophenyl)-2-(4-chlorophenyl)-5-methyl-1-(propan-2-yl)-1H-pyrroleas a pale yellow solid (36.1 g, 96% yield) which was used withoutpurification. LCMS (ESI) [M+H]+=406.1/408.1

Synthesis of1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine

To a solution of3-(3-bromo-5-fluorophenyl)-2-(4-chlorophenyl)-5-methyl-1-(propan-2-yl)-1H-pyrrole(24 g, 59.0 mmol) in anhydrous DMSO (200 mL) was added4-nitrophenylpiperazine (33 g, 159 mmol) and DMPAO (11.6 g, 60 mmol)under nitrogen atmosphere. The mixture was warmed and sonicated toassist with solubilization and nitrogen gas was bubbled through thesolution for several minutes. Potassium carbonate (32.44 g, 234.7 mmol)and copper (I) iodide (2.88 g, 15.1 mmol) were then added and nitrogenbubbling continued for another 5 min. The mixture was heated to 120° C.for 6 h under nitrogen. The mixture was treated with DCM (800 mL), water(1.5 L) and saturated aqueous NH₄Cl (400 mL). The organic layer wasseparated and the aqueous layer extracted with DCM (2×200 mL). Thecombined organic extracts were washed with water (5×500 mL), saturatedaqueous NaHCO₃ (200 mL), water (200 mL) and brine (50 mL), dried overMgSO₄, filtered and concentrated under vacuum.

The crude residue was dissolved in DCM (minimal amount) and treated withMeOH (excess). The mixture was heated to 70° C. until the volume wasreduced by approximately half. The suspension was cooled at 0° C. andthe solid was collected by filtration, washed with cold MeOH and driedunder vacuum to give1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazineas a yellow solid (19.2 g, 61% yield). Additional solid was observed toprecipitate in the filtrate and was collected by filtration, washed withcold MeOH and dried under vacuum to give additional desired product as ayellow solid (1.28 g, 4% yield). LCMS (ESI) single major peak, poorionization

Synthesis of1-[3-[2-(4-chlorophenyl)-4-iodo-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine

To a solution of1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine(20.4 g, 38.3 mmol) in DMF (250 mL) was added a solution ofN-iodosuccinimide (10.45 g, 46.4 mmoL) in DMF (50 mL) at 0° C. Themixture was stirred at room temperature for 2.5 h. The mixture wasdiluted in ice/water (1.3 L). The precipitated solid was collected byfiltration, washed with water then dissolved in DCM. The resultingsolution was dried over MgSO₄, filtered and concentrated under vacuum.The crude residue was triturated with MeOH and the solid was collectedby filtration and dried under vacuum to give1-[3-[2-(4-chlorophenyl)-4-iodo-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazineas a yellow solid (23.0 g, 91% yield) which was used withoutpurification. LCMS (ESI) single major peak, poor ionization

Synthesis of1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-methylsulfonyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine

To a solution of1-[3-[2-(4-chlorophenyl)-4-iodo-1-isopropyl-5-methyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine(13.35 g, 18.23 mmol) in anhydrous DMSO (120 mL) was added prolinesodium salt (3.1 g, 22.3 mmol) and methanesulfinic acid sodium salt(20.2 g, 198 mmol) under nitrogen atmosphere. Copper (I) iodide (3.52 g,18.5 mmol) was then added and the mixture was heated to 105° C. for 19h. The mixture was diluted with EtOAc (1.5 L), water (500 mL) andsaturated aqueous NH₄Cl (300 mL). The organic layer was separated andthe aqueous layer was extracted with EtOAc (500 mL). The combinedorganic extracts were washed with water (2×500 mL), saturated aqueousNaHCO₃ (200 mL), water (200 mL) and brine (200 mL), dried over MgSO₄,filtered and concentrated under vacuum.

The crude residue was purified by silica flash chromatography (0-60%EtOAc in hexanes). Desired product-containing fractions were combinedand concentrated and further purified by recrystallization fromEtOAc/hexanes to give1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-methylsulfonyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazineas an orange solid (9.18 g, 41%). Additional desired product (2.7 g,12%) was obtained via repetition of the process of silica chromatographyand recrystallization from EtOAc/hexanes. LCMS (ESI) [M+H]+=611.2

Synthesis of4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)aniline

To a solution of1-[3-[2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-methylsulfonyl-pyrrol-3-yl]-5-fluoro-phenyl]-4-(4-nitrophenyl)piperazine(2.34 g, 3.82 mmol) in ethanol (50 mL) was added concentratedhydrochloric acid (4 mL) and tin(II) dichloride dihydrate (4.32 g, 19.1mmol) under nitrogen atmosphere. The mixture was heated to reflux for 5h. The mixture was concentrated to ⅓ volume and partitioned betweenethyl acetate (200 mL) and saturated aqueous sodium bicarbonate (200mL). The layers were separated and the aqueous was washed with ethylacetate (2×100 mL). The combined organic extracts were washed with water(3×80 mL) and brine (50 mL), dried over MgSO₄, filtered and concentratedunder vacuum to give4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)anilineas a tan foam solid (2.30 g, >100%) which was used without purification.LCMS (ESI) M+H=581.2

Synthesis of3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)oxazolidin-2-one

To a solution of4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)aniline(581 mg, 1.0 mmol) in anhydrous acetonitrile (20 mL) was added potassiumcarbonate (691 mg, 5.0 mmol, fine powder) under nitrogen atmosphere. Asolution of 2-bromoethyl chloroformate (281 mg, 1.5 mmol) in anhydrousacetonitrile (5 mL) was then added and the resulting mixture was heatedto reflux for 18 h. The mixture was diluted with brine (50 mL) and water(50 mL) and extracted with ethyl acetate (2×50 mL). The combined organicextracts were washed with water (50 mL) then brine (20 mL), dried overMgSO₄, filtered and concentrated under vacuum. The crude residue waspurified by silica flash chromatography (Revelaris, 20 g silicacartridge, 40-100% ethyl acetate in DCM) to provide3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)oxazolidin-2-oneas an off-white solid (365 mg, 58% yield). LCMS (ESI) M+H=651.2,M+Na=673.2

Synthesis of2-((4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)amino)ethan-1-ol

To a solution of3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-Aphenyl)oxazolidin-2-one(365 mg) in ethanol (50 mL) was added a solution of potassium hydroxide(1.5 g, 27 mmol) in water (5 mL). The mixture was heated to reflux for 1h. The mixture was concentrated under vacuum and the residue partitionedbetween ethyl acetate (50 mL) and water (100 mL). The layers wereseparated and the aqueous washed with ethyl acetate (50 mL). Thecombined organic extracts were washed with brine (30 mL), dried overMgSO₄, filtered and concentrated under vacuum to give2-((4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)amino)ethan-1-olas a tan foam (391 mg, 63% yield). LCMS (ESI) M+H=625.3, M+Na=647.2

Synthesis of3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-2-(4-fluoro-3-nitrophenyl)-1,3,2-oxazaphospholidine2-oxide

1^(st) Step

To a suspension of (4-fluoro-3-nitrophenyl)phosphonic acid (pre-driedbefore use by co-evaporation from toluene and drying under high vacuum,1.33 g, 6.0 mmol) in anhydrous DCM (15 mL) was added oxalyl chloride (2mL, 24 mmol) and DMF (2 drops) under a nitrogen atmosphere. After gasevolution had subsided, the mixture was heated to 40° C. for 45 min,cooled and concentrated under vacuum. The resulting residue wascoevaporated from toluene to remove excess oxalyl chloride. The cruderesidue was then redissolved in anhydrous DCM (15 mL) and treated againwith oxalyl chloride (1.5 mL) and DMF (2 drops) and heated to reflux for30 min under nitrogen. The mixture was concentrated under vacuum and theresidue co-evaporated from toluene to give the crude intermediate as anorange oil which was used directly in the next step.

2^(nd) Step

To a solution of2-((4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)amino)ethan-1-ol(2.5 g, 4.0 mmol) in anhydrous DCM (15 mL) and anhydrous pyridine (3.23mL, 40 mmol) was added a solution of the crude residue from the laststep in anhydrous DCM (15 mL) under a nitrogen atmosphere and cooled inan ice/water bath. The resulting mixture was stirred for 30 min thenwarmed to room temperature and stirred for an additional 30 min. Themixture was diluted in DCM (50 mL) and treated with saturated aqueousNaHCO₃ (50 mL). The layers were separated and the aqueous was washedwith DCM (2×50 mL). The combined organic extracts were washed with water(100 mL), dried over MgSO₄, filtered and concentrated under vacuum.

The crude residue was purified by silica flash chromatography(Reveleris, 70-100% DCM in hexanes then 0-100% EtOAc in DCM) to give3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-2-(4-fluoro-3-nitrophenyl)-1,3,2-oxazaphospholidine2-oxide as an orange foam solid (2.23 g, 69% yield). LCMS (ESI)[M+H]+=810.1

Synthesis of3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-2-(4-(((R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)-1,3,2-oxazaphospholidine2-oxide

To a solution of tert-butyl(R)-(4-(dimethylamino)-1-(phenylthio)butan-2-yl)carbamate (120 mg, 0.370mmol) in ethyl acetate (3 mL) was added concentrated aqueoushydrochloric acid (3 mL). The mixture was stirred at room temperaturefor 2 h. The mixture was then concentrated to dryness and the resultingresidue was dissolved in anhydrous DMF (10 mL) under a nitrogenatmosphere. N,N-diisopropylethylamine (485 mg, 3.75 mmol) followed by3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-2-(4-fluoro-3-nitrophenyl)-1,3,2-oxazaphospholidine2-oxide (200 mg, 0.25 mmol) were added and the resulting mixture wasstirred at room temperature for 4 h. The mixture was diluted in brine(50 mL) and water (50 mL) and was extracted with ethyl acetate (2×50mL). The combined organic extracts were washed with water (50 mL) andbrine (20 mL), dried over MgSO₄, filtered and concentrated under vacuum.

The crude residue was purified by silica flash chromatography(Reveleris, 20 g SiO₂ column, 0-10% methanol in DCM) to give3-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-2-(4-(((R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)-1,3,2-oxazaphospholidine2-oxide as a mixture of diastereoisomers and as a yellow foam (157 mg,62% yield). LCMS (ESI) M+H=1014.2, M+2H/2=507.8

Example 10: Biochemical and Cellular Activity of Phospholidine Compounds

Selected compounds were assessed for inhibition of ligand binding toBcl-2 in an in vitro assay, according to the method described inExample 1. The compounds were also assessed for senescent cell killingactivity in HRMEC cell line and IMR90 fibroblasts according to themethod described in Example 3 and 2, respectively.

FIGS. 3A and 3B show the results of both the Bcl binding assay and thecell culture assay.

FIG. 4 is a drawing that depicts a three-dimensional model in which theBcl inhibitors described in this disclosure are fit into the crystalstructure of Bcl family proteins. The annotations in the drawing can beused as a guide to the reader for developing additional compounds thatfall within the formulas shown above, that would retain Bcl inhibitionactivity and senolytic activity. Regions that interact closely with Bclor its binding partners are less tolerant to variation. Regions thatinteract with solvent are more tolerant to variation in terms of Bclinhibition, and can be modified to adjust other properties of themolecule, such as solubility and detection.

The several hypotheses presented in this disclosure provide a premise byway of which the reader may understand various aspects of the invention.This premise is provided for the intellectual enrichment of the reader.Practice of the invention does not require detailed understanding orapplication of the hypothesis. Except where stated otherwise, featuresof the hypothesis presented in this disclosure do not limit applicationor practice of the claimed invention.

For example, except where the elimination of senescent cells isexplicitly required, the compounds may be used for treating theconditions described regardless of their effect on senescent cells.Although many of the senescence-related conditions referred to in thisdisclosure occur predominantly in older patients, the occurrence ofsenescent cells and the pathophysiology they mediate can result fromother events, such as irradiation, other types of tissue damage, othertypes of disease, and genetic abnormalities. The invention may bepracticed on patients of any age having the condition indicated, unlessotherwise explicitly indicated or required.

Discussions about the mechanism of action of the compounds of thedisclosure are also provided for the intellectual enrichment of thereader, and do not imply any limitation. Except where stated otherwise,the compounds may be used for removing senescent or cancer cells or forthe treatment of disease conditions as claimed below, regardless of howthey operate inside the target cells or in the treated subject.

Although the compounds and compositions referred to in this disclosureare illustrated in the context of eliminating senescent cells andtreating senescence-associated conditions and cancer, compounds andtheir derivatives described herein that are novel can be prepared forany purpose, including but not limited to laboratory use, the treatmentof senescence-related conditions, the poisoning of in-laws, and fordiagnostic purposes.

While the invention has been described with reference to the specificexamples and illustrations, changes can be made and equivalents can besubstituted to adapt to a particular context or intended use as a matterof routine development and optimization and within the purview of one ofordinary skill in the art, thereby achieving benefits of the inventionwithout departing from the scope of what is claimed and theirequivalents.

The invention claimed is:
 1. A compound of Formula (I):

wherein: X¹ is O or S; R¹ and R³ together with the N and P atoms throughwhich they are connected form a 5-, 6- or 7-membered heterocyclic ring,optionally substituted with one or more R²³; R⁴ is selected fromhydrogen, alkyl, substituted alkyl, nitro, alkylsulfonyl (e.g.,CH₃SO₂—), substituted alkylsulfonyl (e.g., CF₃SO₂—), alkylsulfinyl,substituted alkylsulfinyl, cyano, C(O)OH, C(O)NH₂, halogen, SO₂NH₂,alkylaminosulfonyl, substituted alkylaminosulfonyl, alkylsulfonylaminoand substituted alkylsulfonylami no, alkanoyl, substituted alkanoyl,alkylaminocarbonyl, substituted alkylaminocarbonyl, alkyloxycarbonyl andsubstituted alkyloxycarbonyl; R²² is selected from hydrogen, alkyl andsubstituted alkyl; each R²³ is independently selected from alkyl,substituted alkyl, —CONH₂, COOH, CONHR²², hydroxyl, halogen, alkoxy andsubstituted alkoxy; Z² is selected from —NR⁵R⁶, hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, alkylsulfanyl, substituted alkylsulfanyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carbocycle, substituted carbocycle, heterocycle, substitutedheterocycle, arylalkoxy, substituted arylalkoxy, aryloxy, substitutedaryloxy, aryloxyalkoxy, substituted aryloxyalkoxy, arylsulfanyl,substituted arylsulfanyl, arylsulfanylalkoxy, substitutedarylsulfanylalkoxy, cycloalkylalkoxy, substituted cycloalkylalkoxy,cycloalkyloxy, substituted cycloalkyloxy, halogen, carbonyloxy,haloalkoxy, haloalkyl, hydroxy and nitro; Z³ is selected fromheterocycle, substituted heterocycle, —NR⁵R⁶, aryl, substituted aryl,heteroaryl, substituted heteroaryl, carbocycle and substitutedcarbocycle; R⁵ and R⁶ are independently selected from hydrogen, alkyland substituted alkyl; R¹¹ and R¹² are each one or more optionalsubstituents each independently selected from alkyl, substituted alkyl,alkoxy, substituted alkoxy, halogen, cyano, nitro, carboxy, C(O)NH₂,SO₂NH₂, sulfonate, hydroxyl, alkylsulfonyl, substituted alkylsulfon24yl,alkylaminosulfonyl, substituted alkylaminosulfonyl, alkylsulfonylamino,substituted alkylsulfonylamino, alkyloxycarbonyl, substitutedalkyloxycarbonyl and —NR⁵R⁶; and R³¹ is selected from H, R¹² and L³-Y³wherein L³ is a linker and Y³ is selected from aryl, substituted aryl,heteroaryl and substituted heteroaryl.
 2. The compound of claim 1, whichhas the structure shown in Formula (IVa) or Formula (IVb):

wherein: Z⁴ is selected from CH, CR¹³ and N; L², L³, L⁴ and L⁵ are eachindependently a linker; Y² is selected from alkyl, substituted alkyl,hydroxyl, alkoxy, substituted alkoxy, —NR⁷R⁸, aryl, substituted aryl,heteroaryl, substituted heteroaryl, carbocycle, substituted carbocycle,heterocycle and substituted heterocycle; Y³ is selected from aryl,substituted aryl, heteroaryl and substituted heteroaryl; Y⁴, Y⁵ and Y⁶are independently selected from aryl, substituted aryl, heteroaryl,substituted heteroaryl, carbocycle, substituted carbocycle, heterocycle,and substituted heterocycle; R⁷ and R⁸ are independently selected fromhydrogen, alkyl and substituted alkyl, or R⁷ and R⁸ together with thenitrogen to which they are attached form a 5-, 6- or 7-memberedheterocyclic ring or substituted 5-, 6- or 7-membered heterocyclic ring;and R¹³ is one or more optional substituents selected from alkyl,substituted alkyl, alkoxy, substituted alkoxy, halogen, cyano, nitro,carboxy, C(O)NH₂, SO₂NH₂, sulfonate, hydroxyl, alkylsulfonyl,substituted alkylsulfonyl, alkylaminosulfonyl, substitutedalkylaminosulfonyl, alkylsulfonylamino, substituted alkylsulfonylamino,alkyloxycarbonyl, substituted alkyloxycarbonyl, alkylamino,dialkylamino, substituted alkylamino and substituted dialkylamino. 3.The compound of claim 2, which has the structure shown in Formula (IVa).4. The compound of claim 2, which has the structure shown in Formula(IVb).
 5. The compound of claim 2, wherein the compound is selectedfrom: i) a compound of Formula (IVa), wherein Y³ is a substituted orunsubstituted fused bicyclic heteroaryl (e.g., a pyrrolo-pyridine); andii) a compound of Formula (IVb), wherein Y³ is a substituted orunsubstituted phenyl.
 6. The compound of claim 2, which has thestructure shown in Formula (V):

wherein: Y² is selected from OR″, OP(═O)(OR″)₂ and NR⁷R⁸; Z⁵ is selectedfrom NR¹⁹, NH, O and S; Z⁷ is selected from S and O; L⁶ and L⁷ areindependently a covalent bond, C₁₋₆alkyl linker or substituted C₁₋₆alkyl linker; R¹³ and R¹⁴ and each R¹⁵ are independently one or moreoptional substituents selected from alkyl, substituted alkyl, alkoxy,substituted alkoxy, halogen, cyano, nitro, carboxy, C(O)NH₂, SO₂NH₂,sulfonate, hydroxyl, alkylsulfonyl, substituted alkylsulfonyl,alkylaminosulfonyl, substituted alkylaminosulfonyl, alkylsulfonylamino,substituted alkylsulfonylamino, alkyloxycarbonyl, substitutedalkyloxycarbonyl, alkylamino, dialkylamino, substituted alkylamino andsubstituted dialkylamino; R¹⁶ is selected from hydrogen, halogen andR¹⁵; R¹⁷ and R¹⁸ are independently selected from hydrogen, cyano, nitro,halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocycle, substituted heterocycle, OR, SR,NRR′, COR, OCOR, CO₂R, CONRR′, CONRSO₂R′, —C₁₋₃ alkyleneCH(OH)CH₂OH,SO₂R and SO₂NRR′; and R¹⁹ is selected from hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,substituted heterocycle, OR, SR, NRR′, COR, OCOR, CO₂R, CONRR′,CONRSO₂R′, NRCOR, NRCONRR′, NRC(═S)NRR′, NRSO₂R and SO₂NRR′; each R″ isindependently H, alkyl or substituted alkyl; and R and R′ areindependently selected from hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,substituted heterocycle, heterocycle-alkyl- and substitutedheterocycle-alkyl-.
 7. The compound of claim 6, which has the structureshown in Formula (VIa):

wherein: Z⁶ is selected from O, NR⁴⁰*, CHR⁴⁰, C(R⁴⁰)₂ and CH₂; R⁴⁰* isselected from alkyl, substituted alkyl, —(CH₂)_(m1)OR and—(CH₂)_(m2)OP(═O)(OR)₂, wherein m1 and m2 are independently an integerfrom 1 to 6 and each R is independently H, alkyl or substituted alkyl;each R⁴⁰ is independently selected from —OR, —N(R)₂, —C(O)OR,—(CH₂)_(m1)OR, —(CH₂)_(m3)N(R)₂, —(CH₂)_(m2)OP(═O)(OR)₂, —OP(═O)(OH)₂,and —OP(═O)(OR)₂ wherein m1, m2 and m3 are each independently an integerfrom 1 to 6 and each R is independently H, alkyl or substituted alkyl;and q₁ and q₂ are independently an integer from 1 to
 6. 8. The compoundof claim 6, which has the structure shown in Formula (VIb):

wherein Y² is selected from OH, OR, NH₂, NHR, NR₂, —OP(═O)(OR)₂ and—OP(═O)(OH)₂ wherein each R is independently C₁₋₆ alkyl or substitutedC₁₋₆ alkyl; and q₁ and q₂ are independently an integer from 1 to
 6. 9.The compound of claim 7, which has the structure shown in Formula(VIIa):

wherein: X² is selected from Z¹², S, O and NR²²; Z¹¹ is selected fromC(═O), —C(═O)X³— and Z¹²; each Z¹² is independently CR²⁴R²⁵; X³ is O orS; n is 0, 1, 2 or 3; each R²⁴ and each R²⁵ are independently selectedfrom hydrogen, alkyl and substituted alkyl; R⁴ is selected from NO₂,SO₂CH₃, SO₂CF₃ and COR⁵¹; Z⁴ is selected from CH and N; Z⁶ is selectedfrom O, CHC(O)R¹⁸, and CH(CH₂)_(p)R¹⁸ wherein p is 0-6 and each R¹⁸ isindependently —OR, —N(R)₂, —OP(═O)(OH)₂, and —OP(═O)(OR)₂ wherein each Ris independently H, alkyl or substituted alkyl (e.g., a C₁₋₄alkyl suchas ethyl or tert-butyl); R¹⁴ and R¹⁶ are independently hydrogen orhalogen; and R¹⁷ is selected from SO₂R⁵², COR⁵², CO₂R⁵², CONR⁵¹R⁵²,CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²; R¹⁸ and R¹⁹ are independently selected fromhydrogen, alkyl and substituted alkyl; R⁵¹ is selected from C₁₋₆alkyland substituted C₁₋₆ alkyl; and R⁵² is selected from hydrogen, C₁₋₆alkyland substituted C₁₋₆alkyl.
 10. The compound of claim 9, selected fromthe following table: Compound X² Z¹¹-(Z¹²)_(n) R⁴ R¹⁴ R¹⁶ R¹⁷ R¹⁸ R¹⁹ Z⁴Z⁶ 1 O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOH 2 O CH₂CH₂ SO₂CF₃ FCl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 3 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃CH(CH₃)₂ N CHOH 4 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 5O CH₂CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 6 O CH₂CH₂CH₂ SO₂CF₃ FCl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 7 O CH₂CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃CH(CH₃)₂ N CHOH 8 O CH₂CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂9 O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 10 O CH₂CH₂ SO₂CH₃ F ClCO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 11 O CH₂CH₂ SO₂CH₃ F Cl SO₂CH₃ CH₃CH(CH₃)₂ N CHOH 12 O CH₂CH₂ SO₂CH₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOPO(OH)₂13 O CH₂CH₂ NO₂ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N CHOH 14 O CH₂CH₂ NO₂ F ClSO₂CH₃ CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 15 O CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂N CHOH 16 O CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOPO(OH)₂ 17 OCH₂CH₂CH₂ NO₂ F Cl CO₂H CH₃ CH(CH₃)₂ N CHOH 18 O CH₂CH₂CH₂ NO₂ F Cl CO₂HCH₃ CH(CH₃)₂ N CHOPO(OH)₂


11. The compound of claim 8, which has the structure shown in Formula(VIIb):

wherein: X² is selected from Z¹², S, O, and NR²²; Z¹¹ is selected from—C(═O)—, —C(═O)X³— and Z¹²; each Z¹² is independently CR²⁴R²⁵; X³ is Oor S; n is 0, 1, 2 or 3; each R²⁴ and each R²⁵ are independentlyselected from hydrogen, alkyl and substituted alkyl; Y² is selected from—OR⁵², —N(R⁵²)₂, and —OP(═O)(OR⁵²)₂; R⁴ is selected from NO₂, SO₂CH₃,SO₂CF₃ and COR⁵¹; Z⁴ is selected from CH and N; R¹⁴ and R¹⁶ areindependently hydrogen or halogen; and R¹⁷ is selected from SO₂R⁵²,COR⁵², 002R⁵², CONR⁵¹R⁵², CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²; R¹⁸ and R¹⁹ areindependently selected from hydrogen, alkyl and substituted alkyl; R⁵¹is selected from C₁₋₆alkyl and substituted C₁₋₆ alkyl; and R⁵² isselected from hydrogen, C₁₋₆alkyl and substituted C₁₋₆alkyl.
 12. Thecompound of claim 11, selected from the following table: Compound X²Z¹¹-(Z¹²)_(n) R⁴ R¹⁴ R¹⁶ R¹⁷ R¹⁸ R¹⁹ Z⁴ Y² 19 O CH₂CH₂ NO₂ F Cl SO₂CH₃CH₃ CH(CH₃)₂ N N(CH₃)₂ 20 O CH₂CH₂ SO₂CF₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ NN(CH₃)₂ 21 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 22 O CH₂CH₂SO₂CF₃ H Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 23 O CH₂CH₂ SO₂CH₃ F Cl CO₂H CH₃CH(CH₃)₂ N N(CH₃)₂ 24 O CH₂CH₂ SO₂CH₃ H Cl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂25 O CH₂CH₂ SO₂CH₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N N(CH₃)₂ 26 O CH₂CH₂ NO₂ FCl CO₂H CH₃ CH(CH₃)₂ N N(CH₃)₂ 27 O CH₂CH₂ NO₂ H Cl CO₂H CH₃ CH(CH₃)₂ NN(CH₃)₂ 28 O CH₂CH₂ NO₂ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N OH 29 O CH₂CH₂ SO₂CF₃F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N OH 30 O CH₂CH₂ SO₂CF₃ F Cl CO₂H CH₃ CH(CH₃)₂N OH 31 O CH₂CH₂ SO₂CF₃ H Cl CO₂H CH₃ CH(CH₃)₂ N OH 32 O CH₂CH₂ SO₂CH₃ FCl CO₂H CH₃ CH(CH₃)₂ N OH 33 O CH₂CH₂ SO₂CH₃ H Cl CO₂H CH₃ CH(CH₃)₂ N OH34 O CH₂CH₂ SO₂CH₃ F Cl SO₂CH₃ CH₃ CH(CH₃)₂ N OH 35 O CH₂CH₂ NO₂ F ClCO₂H CH₃ CH(CH₃)₂ N OH 36 O CH₂CH₂ NO₂ H Cl CO₂H CH₃ CH(CH₃)₂ N OH


13. The compound of claim 1, wherein X¹ is O.
 14. The compound of claim1, wherein X¹ is S.
 15. The compound of claim 9, which has the structureshown in Formula (VIIIIa):

where: R⁴ is selected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹; n is 1 or 2;Z⁶ is selected from O, CHC(O)R¹⁸, and CH(CH₂)_(p)R¹⁸ wherein p is 0-6and each R¹⁸ is independently —OR, —N(R)₂, —OP(═O)(OH)₂, and—OP(═O)(OR)₂ wherein each R is independently H, alkyl or substitutedalkyl (e.g., a C₁₋₄alkyl such as ethyl or tert-butyl); R¹⁴ and R¹⁶ areindependently hydrogen or halogen; and R¹⁷ is selected from SO₂R⁵²,COR⁵², CO₂R⁵², CONR⁵¹R⁵², CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²; R⁵¹ is selectedfrom C₁₋₆alkyl and substituted C₁₋₆ alkyl; and R⁵² is selected fromhydrogen, C₁₋₆alkyl and substituted C₁₋₆alkyl.
 16. The compound of claim11, which has the structure shown in Formula (VIIIb):

where: Y² is selected from —OR⁵², —N(R⁵²)₂, and —OP(═O)(OR⁵²)₂; R⁴ isselected from NO₂, SO₂CH₃, SO₂CF₃ and COR⁵¹; n is 1 or 2; R¹⁴ and R¹⁶are independently hydrogen or halogen; and R¹⁷ is selected from SO₂R⁵²,COR⁵², CO₂R⁵²,CONR⁵¹R⁵², CONR⁵²SO₂R⁵¹ and SO₂NR⁵¹R⁵²; R⁵¹ is selectedfrom C₁₋₆alkyl and substituted C₁₋₆ alkyl; and R⁵² is selected fromhydrogen, C₁₋₆alkyl and substituted C₁₋₆alkyl.
 17. The compoundaccording to claim 1, selected from the following:


18. A phosphorylated form of a compound according to claim
 1. 19. Thecompound of claim 1, which has an E₀₅₀ for irradiated IMR90 cells ofless than 0.1 μM.
 20. A method of selectively removing senescent cellsand/or cancer cells from a mixed cell population or tissue, comprisingcontacting the cell population or the tissue with a compound accordingto claim 1.